Takei Zoological Letters (2021) 7:10 https://doi.org/10.1186/s40851-021-00175-x

REVIEW Open Access The digestive tract as an essential organ for water acquisition in marine teleosts: lessons from eels Yoshio Takei

Abstract Adaptation to a hypertonic marine environment is one of the major topics in physiology research. Marine teleosts lose water osmotically from the gills and compensate for this loss by drinking surrounding seawater and absorbing water from the intestine. This situation is in contrast to that in mammals, which experience a net osmotic loss of water after drinking seawater. Water absorption in fishes is made possible by (1) removal of monovalent ions (desalinization) by the − esophagus, (2) removal of divalent ions as carbonate (Mg/CaCO3) precipitates promoted by HCO3 secretion, and (3) facilitation of NaCl and water absorption from diluted seawater by the intestine using a suite of unique transporters. As a result, 70–85% of ingested seawater is absorbed during its passage through the digestive tract. Thus, the digestive tract is an essential organ for marine teleost survival in the hypertonic seawater environment. The eel is a species that has been frequently used for osmoregulation research in laboratories worldwide. The eel possesses many advantages as an experimental animal for osmoregulation studies, one of which is its outstanding euryhalinity, which enables researchers to examine changes in the structure and function of the digestive tract after direct transfer from freshwater to seawater. In recent years, the molecular mechanisms of ion and water transport across epithelial cells (the transcellular route) and through tight junctions (the paracellular route) have been elucidated for the esophagus and intestine. Thanks to the rapid progress in analytical methods for genome databases on teleosts, including the eel, the molecular identities of transporters, channels, pumps and junctional proteins have been clarified at the isoform level. As 10 y have passed since the previous reviews on this subject, it seems relevant and timely to summarize recent progress in research on the molecular mechanisms of water and ion transport in the digestive tract in eels and to compare the mechanisms with those of other teleosts and mammals from comparative and evolutionary viewpoints. We also propose future directions for this research field to achieve integrative understanding of the role of the digestive tract in adaptation to seawater with regard to pathways/mechanisms including the paracellular route, divalent ion absorption, metabolon formation and cellular trafficking of transporters. Notably, some of these have already attracted practical attention in laboratories.

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Correspondence: [email protected] All transporters have both common names and those derived from the solute carrier (SLC) family. In this review, the SLC family names are shown in addition to the common names at first appearance to indicate similar transport characteristics and susceptibility to common inhibitors. The number after the common name is not hyphenated, and the acronyms are capitalized (e.g., PAT1). Laboratory of Physiology, Department of Marine Bioscience, Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan

© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Takei Zoological Letters (2021) 7:10 Page 2 of 34

(Continued from previous page) Keywords: Osmoregulation, Seawater adaptation, Anguilla spp., Esophageal desalinization, Biomineralization, Epithelial transport, Transcellular transport, Paracellular transport, Tight junction protein, Hormonal regulation, Metabolon, Vesicle trafficking

1. Background ionoregulators and osmoconformers). Thus, they need only 1.1 Diverse body fluid regulation in marine fishes to excrete excess ions and retain urea at the kidneys, as os- Extant vertebrates inhabit three different types of habi- motic forces on water diffusion are nearly abolished. Mar- tats in terms of osmoregulation: water- and ion-deficient ine teleosts use a third strategy like that of marine land habitats, water-sufficient and ion-deficient inland mammals, birds and reptiles, which have plasma ion con- freshwater (FW) habitats, and water- and ion-sufficient centrations and osmolality approximately one-third that of seawater (SW) habitats. Because of the high osmolality SW (making them iono- and osmoregulators) (Fig. 1). of SW, however, the ocean is generally a desiccative en- Thus, they must counter osmotic water loss and excess ion vironment for marine teleost fishes. To address this gain. To address this hydromineral challenge, marine tele- issue, marine fishes use three different strategies for osts drink SW and absorb water together with ions in the body fluid regulation (Fig. 1). The first strategy is intestine. Ions also enter the body via the body surfaces adopted by the most primitive extant vertebrate group, driven by their concentration gradients. Then, excess the hagfishes, which conform their plasma to environ- monovalent ions are excreted mostly by the gills, and excess mental SW in terms of both ion concentrations and divalent ions are excreted by the kidneys [127, 144]. osmolality (acting as iono- and osmoconformers), similar Gill ionocytes (mitochondrion-rich cells) are respon- − to marine invertebrates (Fig. 1). Thus, they exert little sible for active Na+ and Cl excretion (see [36, 51]). As osmoregulation effort and incur little expense. The ions acquisition of ionocytes appeared to be one of the cues − involved are monovalent ions (Na+ and Cl ), as divalent for teleosts to re-enter the marine environment after the 2+ 2− ions (Mg and SO4 ) are maintained at levels lower teleost-specific whole-genome duplication that occurred than those in plasma [63]. The second strategy is ca. 300 mya [91], gill ionocytes have been the most in- employed by elasmobranchs and a lobe-finned bony fish, tensively studied tissue in osmoregulation research. Con- the , whose plasma ion concentrations are sequently, the molecular mechanisms of transcellular lower than the concentrations in SW but whose plasma and paracellular ion transport have been elucidated, and osmolality is maintained at a level similar to that in SW via these mechanisms have been summarized in several re- accumulation of urea in the plasma (acting as views (e.g., [30, 85, 86, 90]). SW contains high

Fig. 1 Diverse strategies for adaptation of fishes to a hypertonic seawater environment. The ion concentrations are in mM, and osmolality (Osm) is in mOsm/l. The values for eels are for eels acclimated to seawater. The values for seawater are shown in Table 1. All marine mammals, birds and reptiles are iono- and osmoregulators, while the only amphibian that can acclimate to seawater, the crab-eating frog (Fejervarya cancrivora), is an ionoregulator and osmoconformer, as are elasmobranchs. Illustrated by Mari Kawaguchi of Sophia University (https://www.ginganet.org/mari) Takei Zoological Letters (2021) 7:10 Page 3 of 34

2+ 2− concentrations of the divalent ions Mg and SO4 in mammals are usually lower than SW NaCl concentra- addition to monovalent ions (Table 1), and the sites of tions, water is further lost in the urine for NaCl excre- MgSO4 excretion are the proximal tubules of the kidneys tion. Although there is a report showing that whale − [18, 19]. Transporters involved in transcellular Mg2+ kidneys can concentrate Cl to a concentration of 820 transport have been suggested to exist in the euryhaline mM [189], urine NaCl concentrations are usually much pufferfish mefugu, obscurus [96, 97], and those lower than SW concentrations [20]. Marine birds and 2− for SO4 transport have been proposed to exist in the reptiles possess salt glands that concentrate NaCl above mefugu [107]andeel[256]. However, the whole picture of SW levels through a mechanism similar to that in teleost divalent ion transport in the kidneys has not yet been re- ionocytes, which are localized in different parts of the vealed. The urinary bladder also serves as a site for final body [189]. water absorption before excretion in marine teleosts [134]. On the other hand, marine teleosts can absorb 70– It has been known since the early 1900s that marine 85% of ingested SW across the intestine and excrete ex- teleosts drink copiously to compensate for water lost os- cess NaCl from gill ionocytes [127, 144]. This is due to motically across the body surface [207]. The mechanisms the excellent ability of the teleost digestive tract to for eliciting drinking have been investigated in various process ingested SW along its segments for final absorp- teleost species and have been summarized in a few re- tion in the intestine (see [69, 259]). As detailed below, views [69, 218, 221, 259]. Relatively recently, the roles of ingested SW is first diluted in the esophagus by removal − cerebral mechanisms regulating drinking (thirst) in tele- of Na+ and Cl (desalinization) without water loss [83, osts were suggested after the mechanisms were com- 166]. The ingested SW is further diluted in the anterior − pared between fully aquatic eels and semiterrestrial intestine by bicarbonate ion (HCO3 ) secretion into the mudskippers [11, 105]. After SW is consumed, it enters lumen, which is followed by reductions in Mg2+ and the digestive tract, which is an intracorporeal but exter- Ca2+ concentrations via precipitation of these ions as nal environment in relation to body fluids. Only after ab- carbonates [70, 73, 264]. Finally, water is absorbed in − sorption by the intestine does the ingested water join the parallel with Na+ and Cl by the intestine, either trans- body fluids. In this sense, intestinal water absorption is cellularly via the suite of transporters unique to marine critical for body fluid regulation and thus for survival of teleosts [69, 133, 259] or paracellularly via tight junc- marine teleosts in hypertonic SW. tions (TJs). Thanks to the recent development of technology 1.2 Role of the digestive tract in adaptation to marine and bioinformatics together with the establishment of environments genome databases for various fish species, it is possible For terrestrial vertebrates such as mammals, drinking to identify transport molecules responsible for ion and SW results in severe loss of body fluids. Water is lost by water absorption along the digestive tract. The progress osmosis in the esophagus and stomach when hypertonic made in this decade is evident: molecular mechanisms SW enters the lumen. NaCl is absorbed significantly by have been elucidated at the isoform (paralog) level for the intestine and NaCl absorption may be accompanied transporters, channels, pumps and TJ proteins. In re- by absorption of some water. However, as Mg2+ and sponse to such progress and because almost 10 y have 2− SO4 in SW are scarcely absorbed by the intestine, the passed since the previous reviews on teleost intestinal luminal fluid osmolality increases after water absorption, function for osmoregulation were published [69, 213, which hinders further water absorption. Thus, severe 259], we decided to summarize current knowledge about diarrhea occurs after SW drinking in mammals. More- the molecular mechanisms in order to gain insights into over, as urine NaCl concentrations of terrestrial the future directions. In this review, we focus on the

Table 1 Ion concentrations and osmolality of luminal fluid along the digestive tract of eel. Ion concentrations of eel plasma and of seawater are also shown + + - - 2+ 2+ 2- Segment Osmolality (mOsm/l) Na (mM) *K (mM) Cl (mM) HCO3 (mM) Mg (mM) Ca (mM) SO4 (mM) Esophagus 449 211 10 225 - 40 8 - Stomach 460 222 14 255 - 41 10 - Intestine 295 40 14 68 100 150 12 133 Rectum 300 4 11 59 105 188 14 105 SW 1029 450 10 524 2 50 10 30 Plasma 377 175 4 155 14 3 2 1 -, not measured. The data are based on Tsukada et al. [241] *Data of Pleuronectes platessa [74] Takei Zoological Letters (2021) 7:10 Page 4 of 34

recently identified transport molecules in relation to the salt and water balance in fish [273]. We can exclude specific function of each segment of the digestive tract. such influences when eels are used as experimental fish. Although molecular studies have been performed in sev- In addition, we can exclude the influences of nutrient- eral teleost species, we describe the mechanisms mainly coupled ion absorption on water and ion balance when in eels (genus Anguilla) for reasons explained in the next unfed eels are used. It is known that ~ 50% of water is section. We include data obtained in mammals wherever absorbed in parallel with Na+-glucose cotransport in the appropriate to compare the mechanisms of water- human intestine [132]. Furthermore, whole-genome se- retaining and ion-excreting marine teleosts with those of quencing of three Anguilla species has been completed, water- and ion-retaining mammals [225]. and the results are publicly available [79, 101, 168]. Thus, mining of transporter at the paralog level is 1.3 Contribution of eels to osmoregulation research possible with the database. In this review, data for eels Among eels in the order Anguilliformes, the eel dis- are primarily introduced with particular emphasis on the cussed here is a euryhaline, migratory (catadromous) molecular mechanism to serve as a basis for comparison species that can readily adapt to both hypotonic and with the molecular mechanisms of other teleosts. This hypertonic environments. Eels are SW species in origin, review updates the previous reviews on the role of the and some eels do not undergo upstream migration but intestine in osmoregulation [69, 213, 259] and extends rather stay in coastal SW areas throughout their lives, as our previous review on the regulation of drinking in shown by the presence of strontium in whole otoliths fishes, i.e., ingestion from the environment into the di- [242]. Thus, they have an excellent hypo-osmoregulation gestive tract [220]. Ion transport by the digestive tract ability and can easily survive in concentrated SW. Ac- significantly affects acid-base balance, which is referred cordingly, they can be used to examine how osmoregula- to only when necessary in this review. Readers can find tory mechanisms are altered after direct transfer from several reviews on this topic elsewhere [76, 232, 272]. A FW to SW seemingly without severe disturbances in detailed account of osmoregulation in invertebrates and other homeostatic mechanisms. For these reasons, the vertebrates can be found in Larsen et al. [127]. European eel (A. anguilla), Japanese eel (A. japonica) and American eel (A. rostrata) have often been used for 2. The esophagus as an organ for desalinization osmoregulation researches since the time of Smith The class Teleostei is the most diverse group of verte- [207]. These researches include research on gill func- brates in terms of ecology and physiology and contains tion [100, 103, 108, 186], drinking regulation [82, 141, more than half of the total number of vertebrate species 161, 223], and digestive tract function [5, 83, 114, [159]. Unsurprisingly, the morphology of the digestive 138, 165, 206, 238]. In contrast, another family of tract is also diverse among teleost species [261]. Most euryhaline migratory (anadromous) species, the teleost digestive tracts include an esophagus, stomach, salmonids, are FW species in origin, and some live intestine, and rectum; some lack the stomach, such as their whole lives in FW (land-locked subspecies). As a that of medaka (Oryzias spp.), and some have pyloric result, the osmoregulatory mechanisms of eels differ caeca at the anterior part of the intestine, as observed in considerably from those of salmonids and from those salmonids. The esophagus is the first segment of the di- of stenohaline sedentary marine teleosts [224]. Thus, gestive tract that directly receives ingested SW. There it is worthwhile to compare the roles of the digestive are sphincters at the entrance and exit of the esophagus tract in SW adaptation among the three groups with to regulate inflow from the buccal cavity and outflow to different osmoregulatory mechanisms. the stomach. Therefore, ingested SW is kept in the Another advantage of the eel as an experimental fish esophagus for some time to allow modifications to its for studying intestinal function is that this fish can sur- composition. The eel is an excellent experimental fish vive normally for several months without food. We accli- with which to study esophageal function because this mated eels in FW aquaria for 1 week after purchase and fish has a long and distensible esophagus that is suitable then transferred them to SW aquaria for 2 weeks to pre- for use in sac experiments [83, 114, 157, 227]. A mor- pare SW-acclimated eels. We used them for intestinal phological study has shown that the eel esophageal epi- experiments thereafter, and we obtained a full response thelium becomes thinner and that blood vessels develop to hormones in terms of sensitivity and efficacy for a few beneath the epithelium after SW acclimation, supporting months thereafter (see Sect. 5), although in killifish the occurrence of facilitated desalinization [275]. (Fundulus heteroclitus), the water permeability of the in- As shown in Table 1, ingested SW is processed grad- testine appears to decrease within 24 h after feeding ually according to its passage through the digestive tract [274]. The major function of the intestine is nutrient of eels [113, 207, 241, 257]. Similar results have been absorption, and ions and water in the food and in the di- reported in the digestive tract of the winter flounder, gestive juice secreted after feeding significantly influence Pseudopleuronectes americanus [166]. In both species, Takei Zoological Letters (2021) 7:10 Page 5 of 34

− profound decreases in Na+ and Cl concentrations (de- database, and real-time qPCR was performed using salinization) occur in the esophagus. In contrast, the de- paralog-specific primers after transfer of eels from FW 2+ 2− creases in Mg and SO4 concentrations are small, to SW [227]. The final candidate transport proteins that suggesting that esophageal epithelia are scarcely perme- met the criteria are shown in Fig. 2 together with the able to divalent ions and water; thus, osmotic influx of changes in the transcript levels after SW acclimation. water from the serosal side is negligible [83]. In vitro studies using esophageal sac preparation have shown Mucosal side that NaCl efflux increases while water influx decreases For transcellular NaCl absorption, the major route on dramatically after SW acclimation in eels when SW is on the mucosal side is via coupled NHE3 (SLC9a3) and AE the luminal side and isotonic Ringer solution is on the on the apical membranes of epithelial cells in exchange + − serosal side [83, 227]. Unidirectional (mucosa-to-serosa) for H and HCO3 excretion into the lumen (Fig. 2). 22Na fluxes have also been examined using esophageal Several AE genes of the SLC26 and SLC4 families are epithelia in Ussing chambers for the winter flounder expressed in the esophagus, of which apically located [166] and the gulf toadfish, Opsanus beta [49]; the SLC26a3a/b (also called downregulated in adenoma, results show that 22Na efflux is elevated in fish kept in DRA) and SLC26a6a (also called putative anion trans- hypersaline media. porter 1, PAT1) are candidates, but their expression − The transporters involved in transepithelial Na+ and Cl levels are low and not upregulated after SW acclimation. transport have been examined using various transporter- DIDS-sensitive SLC4a2a (AE2a) is another candidate, as specific inhibitors (Table 2). Although the efficacy of its gene is abundantly expressed in the eel esophagus as inhibition varies among species, consistent effects are ob- in the mammalian intestine [244]. Both apical and baso- tained with mucosal application of DMA/EIPA and DIDS/ lateral localization of AE2 have been reported in the DNDS and with serosal application of ouabain and DPC, mammalian intestine [174]. NHE3 and SLC26a3/6 may suggesting the involvement of the apical Na+/H+ exchan- be bound together to the scaffolding protein NHERF1 − − ger (NHE) and Cl /HCO3 exchanger (anion exchanger, (NHE regulatory factor 1) via the PDZ-binding motif to − AE) and of basolateral Na+/K+-ATPase (NKA) and Cl form a metabolon (see 6.3). Their activities may be channels (ClCs) [49, 157, 166, 227]. HCTZ is effective in regulated by NHERF1 through phosphorylation and/or the eel [227]butnoteffectiveinthetoadfish[49]. We ini- vesicle trafficking of the complex to the apical mem- tially thought that HCTZ inhibits the SLC12 family of brane (see 6.4). The NHERF1 gene (slc9a3r1)is − transporters, such as the Na+-Cl cotransporter (NCC), expressed significantly in the eel esophagus [227]. As found in the intestine (see 4.4.1), but HCTZ also inhibits various second messengers (cAMP, Ca2+ and cGMP) + − carbonic anhydrase (CA), which supplies H and HCO3 regulate the activity of the metabolon, hormonal regula- for NHE and AE to facilitate their combined activity [205]. tion of esophageal desalinization most likely occurs. The activity of coupled NHE3 and AE should be en- + − 2.1 Molecular mechanisms of desalinization hanced by the production of H and HCO3 from CO2 Transporter molecules involved in desalinization have hydration by cytosolic CAII, which is expressed abun- been assessed by transcriptome analysis (RNA-seq) using dantly in the esophagus in SW eels [227]. CAII is also the esophagi of FW- and SW-acclimated eels [227]. known to bind to NHERF and to form a metabolon for Among the candidates implied by the inhibitor studies, its regulation (see 6.3). Two CAII genes (ca2a and ca2b) the candidates were further narrowed down with criteria are expressed in the eel esophagus, and ca2a is more of (1) sufficient expression in the esophagus and (2) up- abundant and upregulated after SW acclimation (Fig. 2). regulation in SW-acclimated eels. Since RNA-seq can Thus, CAIIa plays a major role in the production of H+ − hardly distinguish the expressed genes at the isoform and HCO3 for activation of NHE3 and AE. This is in level, all paralogs were mined from the eel genome contrast to the eel intestine, where CAIIb is a major

Table 2 Effects of inhibitors on desalinization in the esophagus of teleosts Species Mucosal side Serosal side Reference Amiloride DMA/EIPA DIDS/DNDS Bumet/Furo HCTZ DPC Ouabain DPC (ENaC) (NHE) (AE, NBC) (NKCC) (NCC) (ClC) (NKA) (ClC) Winter flounder + ND ND – ND ND ++ ND [166] Japanese eel ND ND + + + ND ++ ND [157] Gulf toadfish – +NDND– ND ND ND [49] Japanese eel ND ++ + – ++ – ++ ++ [227] +, effective; −, ineffective. Number of + indicates strength of the effect. In parenthesis is target transporter of the inhibitor. For details, see text and abbreviation list. Bumet bumetanide, Furo furosemide, ND not determined Takei Zoological Letters (2021) 7:10 Page 6 of 34

Fig. 2 Major transporters responsible for desalinization (NaCl absorption) and low water permeability in the esophageal epithelia of SW eels. CA + − provides H and HCO3 to facilitate coordinated action of NHE and AE for NaCl absorption. Upregulation of the genes is shown in the inserted figures. The molecular identity of AE is still unknown because of the lack of upregulated genes in SW. AQP genes are downregulated in SW. For details, see 2.1. For abbreviation definitions, see the list. Modified from Takei et al. [227]

− + cytosolic CAII that produces HCO3 for its secretion responsible for Na uptake given the reduced expression into the lumen and carbonate precipitation (see 4.4.1 of the NHE3 gene in concentrated SW. On the other and 4.4.2). hand, nhe2 is constitutively expressed in the esophagus In the gulf toadfish, the NHE1 (SLC9a1), NHE2 in both FW and SW eels. Thus, NHE2 may also function (SLC9a2), NHE3, CAII, SLC26a6 and AE2 genes are to take up Na+ irrespective of environmental salinity, expressed in the esophagus, but none of the genes are while upregulated NHE3 is specifically involved in the upregulated after transfer of the fish from SW to con- enhanced Na+ uptake in the SW eel esophagus. Recruit- centrated 60 ppt SW [49]. EIPA inhibits desalinization, ment of NHE3-tagged vesicles to the apical mem- and among EIPA-sensitive NHEs, NHE2 is likely brane has been reported to occur in response to Takei Zoological Letters (2021) 7:10 Page 7 of 34

stimuli via intracellular messengers in mammals (see 2.3 Paracellular pathway 6.4). The differences in the major transporters used The TJ protein genes cldn1, cldn3b, cldn5b, cldn7b, for esophageal desalinization may reflect the differ- cldn11b, cldn11b, cldn12, cldn15 and cldn23a are ences in osmoregulatory mechanisms among euryhal- expressed in the esophagus, as detected by RNA-seq, of ine migratory eels and stenohaline, sedentary marine which cldn3b, cldn5b, and cldn15 are upregulated in toadfish [224]. SW-acclimated eels [227]. TJs are composed of proteins from the claudin (CLDN) family and of TJ-associated MARVEL proteins such as occludin and tricellulin, but Serosal side CLDNs are the primary proteins that determine para- − On the serosal side of epithelial cells, Na+ and Cl that cellular ion and water permeability [78]. Among the enter the cells are extruded into the extracellular expressed CLDN genes, cldn15a is exceptionally highly interstitial fluid by the coordinated action of ouabain- expressed, and its expression is profoundly upregulated sensitive NKA (NKA1c1 and NKA3) and DPC- (> 80-fold) after SW acclimation, as detected by RNA-seq; − sensitive Cl channel 2 (ClC2) on the basolateral this upregulation has been confirmed by qPCR (Fig. 2). As membrane (Fig. 2). NKA1c1 and NKA3 are catalytic CLDN15 is known to act as a cation channel in mammals α-subunits of NKA, and these genes (atp1a1c1 and [121], it may serve not only as a barrier for water move- atp1a3) are profoundly upregulated after SW acclima- ment but also as a paracellular route for Na+ uptake (Fig. tion (Fig. 2). The NKA1c2 and NKA1c3 genes are also 2)ifitisalsoNa+-permeable in teleosts (see 6.1). The expressed significantly in the esophagus, but no upreg- same sets of CLDN genes are expressed in the esophagus ulation occurs after SW acclimation [227]. Thus, these and intestine except that cldn3b is additionally expressed NKAs may play a maintenance role in Na+ absorption in the esophagus. All CLDNs expressed in the esophagus in the esophagus in both FW and SW eels. Eels have other than CLDN15 are barrier-forming CLDNs [121], no NKA1a and NKA1b subunits like those found in and the amounts of all transcripts are much greater (~ 10- salmonid gills [85], but the NKA1c subunit is present fold) in the esophagus than in the intestine. Thus, these and diversified into 3 isoforms in eels [270]. The K+- CLDNs form a barrier and inhibit paracellular water − Cl cotransporter 1 (KCC1, SLC12a4) gene is also transport in the SW eel esophagus (Fig. 2). Paracellular expressed significantly in both the FW and SW eel tightness can also be confirmed by the much higher trans- − esophagus; this gene may be responsible for Cl extru- epithelial resistance of the esophagus than of the intestine sion and recycling of K+ accumulated by NKA in the in SW eels. cell on the serosal side. It is also possible that AE2 is − − involved in Cl extrusion in exchange for HCO3 ,asis 3.1 Do the stomach and pyloric caeca play roles suggested to occur in the toadfish [49], as both apical in SW acclimation? and basolateral localization has been reported for There is a sphincter at the exit of the stomach that mammalian SLC4-type AEs [174]. keeps SW for further dilution in the stomach for some time before it is sent out to the anterior intes- tine [261]. The resultant stomach expansion inhibits 2.2 Water transport further drinking in the eel [82]. Measurement of the Another feature of the SW eel esophagus is low water drinking rate in the eel by the esophageal fistula permeability [83]. The low water permeability through method with reintroduction of ingested water into the the epithelial cells is accounted for by the low expres- stomach [226] has revealed that SW eels do not drink sion levels of the two aquaporin genes aqp1a and constantly but rather drink in a rhythmic pattern, i.e., aqp3, which are further downregulated after SW ac- with repeated increases and decreases in intervals of climation (Fig. 2). AQP1 may be on the apical mem- 15–30 min. This indicates that the sphincter is relaxed branes of epithelial cells, and AQP3 may be on the after the interval, which relieves stomach expansion basolateral membrane, as suggested by the localization and restores vigorous drinking in SW. of these AQPs in the intestine (see 4.2.1). The In some species, such as salmonids and sparids, diver- amount of aqp1a transcripts in the esophagus is 1/50 ticula grow from the anterior intestine to form small that in the intestine. On the other hand, aqp1dup, blind-end tubes known as pyloric caeca. The number of most likely aqp1b, is expressed weakly in the esopha- pyloric caeca varies among species, ranging from 5 to 6 gus in European eels and upregulated after cortisol in sea bream to > 200 in salmonids. The function of pyl- treatment [149]. Plasma cortisol concentrations in- oric caeca has attracted attention since Aristotle’s era, crease transiently after SW transfer, and cortisol ad- anditwasshownthatthistissueservesasanexten- ministration increases intestinal water absorption in sionoftheintestinetoincreasethesurfaceareafor eels [84]. nutrient absorption [25]. Vigorous water absorption Takei Zoological Letters (2021) 7:10 Page 8 of 34

has been demonstrated to occur in the pyloric caeca of electroneutral [164]. Furthermore, NaCl absorption is the chinook salmon, Oncorhynchus tshawytscha, and the completely blocked by mucosal application of furosem- uptake capacity is sixfold higher than that of the intestine ide or bumetanide, each of which is an NKCC inhibitor, in SW-acclimated fish [250]. In the gilthead sea bream, in the intestines of SW-acclimated eels [7, 12]; red drum, Sparus aurata, isolated enterocytes from pyloric caeca Sciaenops ocellatus [50]; and other marine teleosts (see possess higher NKA activity than those from the intestine [69, 133]). These results suggest that the major trans- [45]. High NKA activity has also been found in the pyloric porter on the apical membrane responsible for NaCl ab- caeca of chinook salmon, and cortisol treatment further sorption is an NKCC, probably NKCC2. As NKCC2 augments NKA activity and the capacity for water absorp- takes up four ions (osmolytes) into the epithelial cell at a tion [248]. In addition to NKA, high expression of aqp8b time, water efficiently moves in parallel into the cell has been found in the pyloric caeca of the Atlantic salmon, through AQPs (see 4.2). Mucosal application of Ba2+,a Salmo salar, supporting the role of water absorption from K+ channel inhibitor, also blocks NaCl absorption so imbibed SW in this tissue [235]. Epithelial conductance is that recycling of K+ back into the lumen via a K+ chan- higher in the pyloric caeca than in the anterior intestine in nel is necessary for continuous functioning of NKCC2 the rainbow trout Oncorhynchus mykiss [75], suggesting [57]. A shortage of K+ in the lumen easily develops be- that precipitation of divalent ions could occur not only in cause of the low concentration of K+ in SW compared − the intestine but also in the pyloric caeca (see 4.4). The with the concentrations of Na+ and Cl (Table 1). K+ ef- − pyloric caeca may perform both desalinization and water flux at the mucosal side and Cl efflux at the serosal side absorption, but further studies are required to clarify the produce a serosa-negative transepithelial potential differ- osmoregulatory roles of this intriguing tissue. ence (PD), which is characteristic of the marine teleost intestine (Fig. 3). In mammalian intestines, dominant 4. The intestine is an essential organ for water Na+ influx at the mucosal side by epithelial Na+ chan- acquisition nels (ENaCs) produces serosa-positive PD [106]. In the After desalinization in the esophagus and subsequent distal intestine, including the rectum, NCC, more accur- minor processing in the stomach, ingested SW becomes ately NCC1 (SLC12a3), is also involved in NaCl absorp- only slightly hypertonic to body fluids when it enters the tion in the eel intestine (see 4.1.1). − intestine. The major task of the intestine for osmoregu- As observed in the esophagus, Cl is also taken up by − lation is to absorb water together with NaCl using a AE in exchange for HCO3 [68, 69]. This mechanism − − suite of transporters, of which Na+-K+-2Cl cotranspor- is supported by the fact that removal of Cl from the ter 2 (NKCC2, SLC12a1) is unique to marine teleosts mucosal fluid inhibits NaCl absorption (which also in- (see 4.1). As SW contains high concentrations of diva- hibits NKCC2) and the fact that AE is responsible for − lent ions (Table 1) and as these ions are hardly absorbed HCO3 secretion into the lumen for carbonate precipi- by the intestine [166, 207], water absorption results in tation (see 4.4). However, mucosal application of increased divalent ion concentrations in the luminal DIDS, an AE inhibitor, minimally inhibits NaCl ab- fluid. As 74–85% of water is absorbed by the intestine in sorption in the intestine in SW-acclimated eels [7]and 2+ 2− SW eels [207], the concentrations of Mg and SO4 in the goby Gillichthys mirabilis [43]. In addition, more − the luminal fluid become 250 mM and 150 mM, respect- than 50% of Cl absorption is accounted for by AE ively, if 80% of the water is absorbed from SW. As a re- function in the toadfish and some other marine tele- sult, the osmolality produced only by these ions almost osts [68, 73]. To complete NaCl uptake by AE, Na+ is equals that of plasma, although the osmotic coefficient takenupinexchangeforH+ by NHE, as observed in of MgSO4 (0.58) is lower than that of NaCl (0.93). The the esophageal epithelium (Fig. 2) and in the intestines high osmolality produced by the divalent ions certainly of mammals (see 4.1.1). Collectively, the major apical inhibits additional water absorption. To overcome this transporters responsible for NaCl absorption are − problem, the marine teleost intestine secretes HCO3 NKCC2 in the intestines of SW eels and other marine into the lumen and decreases Ca2+ and Mg2+ concentra- (euryhaline) teleosts, while the combination of AE and tions via precipitation of these ions as carbonates (see NHEplaysasignificantroleinsomemarinespecies. 2+ 2− 4.4). In addition, Mg and SO4 appear to be absorbed This variation in responsible transporters also illus- meaningfully by the intestine, as discussed in 6.2. trates the diversity of osmoregulatory mechanisms among teleost species [224]. 4.1 NaCl absorption On the serosal side of the intestinal epithelium, oua- Initial studies have shown that K+ in the luminal fluid is bain, an NKA inhibitor, has been found to significantly essential for NaCl absorption in the intestine in winter block NaCl absorption in all teleost species examined flounder [156] and SW-acclimated eels [6]. The stoichi- thus far (see [69, 133]). Thus, a low cytoplasmic Na+ − ometry of absorbed ions is 1Na+:1K+: 2Cl and thus concentration produced by NKA is essential for the Takei Zoological Letters (2021) 7:10 Page 9 of 34

Fig. 3 Major transporters responsible for Na+ and Cl− absorption (blue rectangles) and water absorption (red rectangle) in the intestinal epithelia of SW eels. K+ extrusion at the mucosal side and Cl− extrusion at the serosal side (black rectangles) produce the serosa-negative potential difference typical of the marine teleost intestine. For the molecular identities of NHE and AE, see Fig. 6. The local increase in osmolality in the lateral interspace (LIS) produced by NKA stimulates water flux into the space through the AQP and tight junction (TJ). The flows of ions and water within the cell are shown by arrows. The text size for the transporters indicates the relative abundance and upregulation of the transporters in SW. For details, see 4.1.1. For abbreviation definitions, see the list

activity of NKCC2, NCC1 and NHE and important not of KCC on the basolateral membrane, which extruces − - + only for NaCl absorption but also for HCO3 secretion Cl and K to the extracellular fluid at the same time, [71]. NKA is an electrogenic pump that extrudes 3Na+ has also been suggested [208]. − in exchange for 2 K+ across the basolateral membrane. Na+ and Cl are also transported via the paracellular As a result, the resting membrane potential of entero- pathway of the intestinal epithelium, which will be dis- cytes becomes negative, and K+ ia maintained in the cussed in detail in section 6.1. The intestinal epithelia of cytosol. The accumulated K+ is recycled into the extra- marine teleosts are leaky and exhibit low transepithelial cellular space via a K+ channel for continuous function- resistance (Rt), and the Rt increases from the anterior to ing of NKA, as the K+ concentration in teleost plasma is posterior direction and is highest in the rectum in the − in the low millimolar range (Fig. 1). Cl channels have eel [12] and in other teleosts [133]. been suggested to be present on the basolateral mem- − brane for Cl efflux into the extracellular space [135]to 4.1.1 Molecular mechanisms of NaCl absorption complete transcellular NaCl transport with NKA. This − Cl efflux is the cause of serosa-negative PD in the mar- Mucosal side Although only one NKCC2 exists in ine teleost intestine, as mentioned above. The presence mammals, two isoforms, NKCC2a and NKCC2b, exist in Takei Zoological Letters (2021) 7:10 Page 10 of 34

the eel [37, 255], of which NKCC2b is the major isoform SW eel gills [216]. In fact, two Kir5.1 genes (kcnj16a and in the intestine (Fig. 3). Of the two NKCC2 genes, kcnj16b) are expressed in the eel intestine, and kcnj16b slc12a1b is expressed at a much higher level than is upregulated in all intestinal segments of SW- slc12a1a and is upregulated in all intestinal segments in acclimated eels (Fig. 4). In the small intestines of mam- eels after acclimation to SW (Fig. 4): [12, 268]. The up- mals, Kir7.1 is involved in recycling of K+ for continuous regulation of slc12a1b occurs after transfer to SW or activity of NKA [167], while Kir5.1 and Kir4.1 are re- hypertonic medium in the intestines of all euryhaline sponsible for K+ recycling for NKA activity in the distal and marine teleost species examined thus far, including convoluted tubules of the kidneys [258]. − the Mozambique tilapia, Oreochromis mossambicus Concerning Cl efflux on the serosal side, ClC2 and [130]; olive flounder, Paralichthys olivaceus [111]; gilt- ClC7 appear to be responsible (Fig. 3) because their head sea bream [66]; red drum [50]; and spotted sea genes (clcn2 and clcn7) are expressed in the eel intestine bass, Lateolabrax maculatus [282]. The slc12a1b expres- and upregulated in the anterior segment after SW accli- sion decreases gradually in the posterior direction in the mation (unpublished data). The clcn2 expression is par- intestine, while the NCC1 gene slc12a3 expression in- ticularly substantial and profoundly upregulated in SW. creases gradually and is highest in the rectum in both The clcn3 is also expressed in the intestine, but its ex- European and Japanese eels [38, 255]. Thus, NCC1 may pression is much lower than that of clcn2 and does not play a role in NaCl absorption in the rectum. However, increase after SW acclimation. In mammals, ClC2 is re- − slc12a3 expression is lower in SW eels than in FW eels, sponsible for Cl efflux at the serosal side of the intes- and it is much lower than that of slc12a1b even in the tinal epithelium [170], but the cell polarity of posterior intestine (Fig. 4). The expression of slc12a3 is localization changes depending on various factors (see also much lower than that of slc12a1 in the intestines 4.3.1). Notably, ClC2 is localized on the lateral mem- of Mozambique tilapia [130] and the three-spine brane close to the TJ and regulates the paracellular per- stickleback, Gasterosteus aculeatus [140]. On the other meability of ions and water in the intestinal epithelium hand, the NCC2 gene (slc12a10) is significantly in mammals [160]. In addition, the KCC1 gene (slc12a4) expressed in the eel intestine, and the expression in- is expressed in the eel intestine, and its expression tends creases in the posterior direction (Fig. 4). NCC2 is in- to increase after SW acclimation (Fig. 4). It seems likely, volved in NaCl absorption in the gills of Mozambique therefore, that KCC1 on the basolateral membrane is re- − tilapia and killifish [224] and probably in the NCC cells sponsible for K+ recycling for NKA and Cl efflux into of the zebrafish, Danio rerio [90]. the interstitial fluid (Fig. 3). Notably, the suite of trans- As mentioned above, active NKCC2 on the apical porters for NaCl absorption develops in the intestine membrane causes K+ shortage in the luminal fluid, during smoltification in the Atlantic salmon, when the which inhibits continuous NKCC2 function. To com- fish are still in FW but preparing for downstream migra- pensate for K+ in the luminal fluid, K+ is recycled by a tion to the sea [215]. K+ channel, and Kir2.1 is the candidate (Fig. 3). The Kir2.1 gene (kcnj2) is expressed substantially in the eel Knowledge from mammals In mammals, the major intestine and upregulated after SW acclimation (Fig. 4). routes for NaCl absorption are (1) electroneutral absorp- − Na+ and Cl are also absorbed from the lumen into tion via combined activity of NHE and AE and (2) elec- enterocytes via the coordinated action of AE and NHE. trogenic absorption via ENaC at the mucosal side of the The molecular identities of AE and NHE are discussed intestinal epithelium [61, 106, 199]. ENaC transports − + in the HCO3 secretion section (see 4.4). only Na , which is the major source of serosa-positive PD in the mammalian intestine. The ortholog of ENaC Serosal side Concerning basolateral transporters for is not identifiable in the teleost genome. Instead, a mem- NaCl absorption, NKA1c1 (atp1a1c1), NKA1c3 ber of the acid-sensing ion channel (ASIC) family, a sub- (atp1a1c3) and NKA1c3 (atp1a3) are responsible for family of the ENaC/degenerin superfamily, exists in Na+ absorption, as they are expressed at considerable teleosts. ASIC, which is localized on the apical mem- levels in the eel intestine [270]. The atp1a1c1 and branes of ionocytes in FW trout gills, takes up Na+ from atp1a3 expression are upregulated in the anterior intes- environmental FW in exchange for H+ through tine after SW acclimation (Fig. 4). These NKAs may vacuolar-type H+-ATPase (VHA) [46]. The transcript of work together to extrude Na+ into the interstitial fluid in ASIC is not detectable in the intestines of eels even by SW eels (Fig. 3). transcriptome analysis (unpublished data). To ensure the constant activity of NKA, K+ should be Two NHE genes (NHE2 and NHE3) are expressed in recycled into the extracellular fluid via a basolateral K+ the mammalian intestine, of which NHE3 is dominant, − − channel, and Kir5.1 is the candidate (Fig. 3). Kir5.1 was and Nhe3 / mice exhibit decreased NaCl absorption first identified as a partner of NKA in the ionocytes of and mild diarrhea [192]. In addition, only NHE3, not Takei Zoological Letters (2021) 7:10 Page 11 of 34

A. Apical NaCl absorption

slc12a1a slc12a1b slc12a3 0.010 3 *** 0.03 0.008 2 0.02 0.006 ** 0.004 1 0.01 0.002

0.000 0 0.00 AI MI PI AI MI PI AI MI PI

slc12a10 kcnj2 1.5 200 * 150 1.0 100 0.5 (relative to ef1a) 50 *

0.0 Reads per million 0 AI MI PI AI PI

B. Basolateral NaCl absorption

atp1a1c1 atp1a1c3 atp1a3 2.0 *** 0.10 0.005

0.08 0.004 1.5 * 0.06 0.003 1.0 0.04 0.002 0.5 0.02 0.001

0.0 0.00 0.000 AI MI PI AI MI PI AI MI PI

kcnj16a kcnj16b slc12a4 0.025 0.008 * 0.004 0.020 0.006 ** 0.003 0.015 ** 0.004 0.002 0.010 0.002 0.001

Gene expression (relative to ef1a) 0.005

0.000 0.000 0.000 AI MI PI AI MI PI AI MI PI

C. Water absorption

aqp1 aqp3 n 0.8 *** 0.004 *** sio

s 0.6 0.003

** xpre 0.4 0.002 e

e 0.2 0.001 en

G 0.0 0.000 AI MI PI AI MI PI

aqp8a aqp10b aqp11 on

i 15000 1000 150 l l i 800 m * 10000 100 600 per

s 400 5000 50 ad 200 * Re 0 0 0 AI PI AI PI AI PI

D. Cl secretion cftra cftrb slc12a2a 0.015 0.0005 0.004

0.0004 0.003 0.010 0.0003 0.002 0.0002 0.005 0.001 * 0.0001

Gene expression 0.000 0.0000 0.000 AI MI PI AI MI PI AI MI PI Fig. 4 (See legend on next page.) Takei Zoological Letters (2021) 7:10 Page 12 of 34

(See figure on previous page.) Fig. 4 Expression of the genes responsible for NaCl (A and B) and water absorption (C) in the anterior intestine (AI), middle intestine (MI) and posterior intestine (PI) in FW-acclimated (plain column) and SW-acclimated (filled column) eels as determined by real-time qPCR. The expression of the genes related to Cl− secretion suggested in mammals and other marine teleosts is also shown in (D). The expression levels are corrected by those of elf1a and thus indicate the relative abundance values of the genes. The atp1, kcnj2 and kcnj16 are the genes for NKA, Kir2.1 and Kir5.1, respectively. The figures with ‘Reads per million’ on the ordinate were created from RNA-seq data (n = 5). The figures are depicted based on the data in Ando et al. [12], Wong et al. [270], and Takei et al. [228] and on unpublished data. The primers for real-time PCR, including those for the unpublished data, are listed in Supplementary Table 1.*p < 0.05, **p < 0.01, ***p < 0.001. For details, see 4.1.1 and 4.2.1. For abbreviation definitions, see the list

NHE2, responds to intracellular messengers such as possible routes for water absorption. As the plasma cAMP for recruitment of transporter-tagged vesicles to membrane consists of a lipid bilayer that is almost im- the apical membrane (see 6.4), which demonstrates a permeable to water, AQPs on the apical and basolateral regulatory role of NHE3 [44]. The major AE genes membranes greatly facilitate transcellular water absorp- expressed in the intestinal epithelium are Slc26a3 and tion [95]. The possibility of water cotransport by trans- Slc26a6. Slc26a3 is expressed throughout the whole porters such as KCC and the Na+-glucose cotransporter length of the intestine, while the Slc26a6 transcript is has been suggested [281], but this idea is still under de- not detectable in the distal colon [106]. It has been sug- bate. Regarding the paracellular route, it is known that − − gested that SLC26a3 exchanges 2Cl /1HCO3 and that some TJ proteins have channel-like activity [121], and − − SLC26a6 exchanges 1Cl /2HCO3 in culture cells with CLDN2 has been suggested to serve as a water channel transient expression, but the stoichiometry is still under (see 6.1). However, paracellular water flux seems to be debate [195]. SLC26a3 plays a major role in NaCl ab- minor in the intestine of the killifish [274]. − − sorption, as Slc26a3 / mice suffer from severe chloride- The marine teleost intestine can absorb water from losing diarrhea [193]. The effect of Slc26a6 knockout on slightly hypertonic luminal fluid [62, 68, 194], which is the intestine is not as strong as the effect on the kidneys also true in SW-acclimated eels [206]. However, a high [200]. NHE and AE form a metabolon with other trans- MgSO4 concentration in the luminal fluid certainly porters and , as discussed in 6.3. limits water absorption and survival in the hypersaline The transporters involved in intestinal NaCl absorp- environment in the toadfish [62]. The luminal fluid close tion in mammals are similar to those of some marine to the apical membrane exists in a microenvironment teleosts but different from those of other teleosts, in- covered by mucus and thus may be made hypotonic to cluding eels, in which NKCC2 plays a dominant role the body fluid by H2O formation and carbonate precipi- (Fig. 3). Interestingly, NKCC2-based NaCl absorption is tation catalyzed by extracellular CAIV (see 4.4.2). On similar to the absorption in the thick ascending limb of the basolateral side, NKA extrudes 3Na+ in exchange for Henle’s loop (TAL) in the mammalian kidneys, in which 2K+, which generates an osmotic gradient between the NKCC2 and renal outer medullary potassium channel intracellular fluid and extracellular fluid. If this occurs in (ROMK) on the apical membrane and NKA, ClC-Kb the lateral space between the adjacent enterocytes just and KCC4 on the basolateral membrane are involved in below the TJ (called the lateral interspace, LIS), the fluid transcellular NaCl absorption [16]. The advantage of the in the LIS becomes hypertonic to both the luminal and NKCC2-based method in teleosts is the efficient trans- intracellular fluids. Then, water moves from both com- port of NaCl and water, as mentioned above, but partments into the interstitial fluid passively [42, 126]. NKCC2 has no regulatory role in acid-base balance, un- Although ion fluxes precede that of water to produce a like the combination of AE and NHE. It is possible that driving force, the fluid transported across the epithelium teleosts acquired the NKCC2 system in the intestine is isotonic, which generates the solute (Na+)-recircula- during the evolution of their habitat toward the ocean; tion model [125]. It is now known that Na+ recirculation this is particularly applicable to migratory/euryhaline is achieved by NHE on the basolateral membrane, not fishes, which must cope with abrupt changes in environ- NKCC1, in the eel (see 4.4.1). The mechanisms of water mental salinity. absorption across intestinal epithelia have been dis- cussed in detail by Whittamore [259]. 4.2 Water absorption In parallel with NaCl transport, water moves from the 4.2.1 Molecular mechanisms of transcellular water intestinal lumen into the body fluid through the epithe- absorption lium if the luminal fluid is almost isotonic to the body Transcellular water movement is achieved through fluid [259]. The transcellular and paracellular routes are AQPs. At least four AQPs are expressed in the eel Takei Zoological Letters (2021) 7:10 Page 13 of 34

intestine, AQP1, AQP3, AQP8, and AQP10 [13, 110, sea bass [64]. Among AQPs expressed in the eel intes- 149], of which AQP3 and AQP10 are glyceroaquaporins tine, AQP3 is the only AQP that has been found to that allow passage not only of water but also of glycerol, localize on the basolateral membranes of epithelial cells urea, etc. [95]. Two isoforms usually exist for AQP1 [39, 254], although AQP3 also localizes to the apical (AQP1a and AQP1b) in the eel and other teleosts. The membrane in killifish [181]. In mammals, apical expression of aqp1a has been found to be upregulated in AQP2 and basolateral AQP3 are responsible for all segments of the eel intestine [13] and in vascular transcellular water absorption at the renal collecting endothelial cells [149]. The tissue distribution of aqp1b duct [102]. transcripts has not yet been examined in eels. The aqp1 AQP8 is another candidate apical membrane AQP expression is most abundant in the rectum, where water (Fig. 3). The aqp8 is expressed in the eel intestine, and absorption may be enhanced by increased hydrostatic the transcript levels increase after SW acclimation [110, pressure [109]. A valve-like structure exists between the 149]. We have also observed upregulation of aqp8a in posterior intestine and the rectum in eels, which blocks the posterior intestines of SW eels (Fig. 4). Immunohis- backflow when the luminal pressure in the rectum in- tochemical analysis showed that AQP8 exists on the ap- creases. AQP1 is localized on the apical membranes of ical membranes of intestinal cells. Three AQP8 genes intestinal epithelial cells, as indicated by immunohisto- (aqp8aa, aqp8ab and aqp8b) are expressed in the intes- chemistry of the intestines of SW-acclimated Japanese tine in Atlantic salmon, of which aqp8ab has the highest and European eels [13, 149]. Our recent study using transcript levels in the intestine, and aqp8ab is upregu- transcriptome analysis (RNA-seq) showed that aqp1a, lated after SW acclimation [236]. The salmon AQP8ab is aqp3, aqp8a, aqp10b and aqp11 were expressed in the also localized on the mucosal (apical) side in enterocytes eel intestine, of which aqp1a and aqp8a were expressed and appears to play important roles in water absorption at high levels (Fig. 4). Isoforms were identified by a [47], as is the case for mammalian AQP8 [240]. reverse-BLAST best hit approach in our eel transcrip- The aqp10 expression is observed in the intestines of tome data (DDBJ accession number: DRA004258) using eels and is upregulated in SW-acclimated fish [110, 149]. annotations in the zebrafish database (https://zfin.org/). However, our transcriptome data suggest that aqp10b is From the RNA-seq data, the expression of aqp1a, aqp3 downregulated in the SW eel intestine (Fig. 4). and aqp8a was significantly upregulated after SW accli- The aqp10 expression has also been reported in the mation, but real-time qPCR showed that aqp3 expres- intestines of Atlantic salmon [236] and gilthead sea sion did not change after SW acclimation (Fig. 4), bream [185]. Cellular localization of AQP10 has not suggesting the possible presence of isoforms. On the yet been examined in fish, but AQP10 is localized on other hand, our qPCR analysis showed that aqp1a was the apical membranes of enterocytes in humans [154]. expressed in all intestinal segments and profoundly up- Expression of aqp3, aqp8 and aqp10, including their regulated after SW acclimation in the eel (Fig. 4). subtypes, has also been reported in the intestine of In the Atlantic salmon [236] and gilthead sea bream the three-spine stickleback [140]. [172], both aqp1a and aqp1b are expressed in the intes- To summarize the possible transcellular pathway for tine, with aqp1a exhibiting higher expression than water absorption, the major apical AQP may be AQP1a aqp1b, and the expression of these genes is higher in fish in the eel intestine, as judged by the expression level of acclimated to SW than in those acclimated to FW. Im- its gene and upregulation after SW acclimation (Fig. 3). munoreactive AQP1a is localized on both the apical and In other marine teleosts, AQP8 and AQP10 on the ap- lateral membranes of sea bream enterocytes, while it is ical membrane are also involved in the uptake of water found on the basolateral membrane in the Atlantic into epithelial cells. The water taken up into the cells salmon. It has been suggested that the direction of may be transported into the extracellular fluid via AQP3 trafficking of transporter-tagged vesicles to either the on the basolateral membrane, although AQP3 gene ex- apical or basolateral membrane is dependent on en- pression is low compared with that of other AQPs. It vironmental salinity (see 6.4). Upregulation of aqp1 seems that AQP1 also plays a role in water absorption at has also been reported in the intestine of the silver the serosal side in the Atlantic salmon [236]. In the sea bream, Sparus sarba [40], and the European sea euryhaline medaka, Oryzias latipes, aqp1a, aqp7, aqp8ab bass, Dicentrarchus labrax [64]. and aqp10a are downregulated after SW transfer, and The aqp3 is most abundantly expressed in the gills of immunoreactive AQP1a and AQP10a move from the ap- teleost fishes, but significant expression has also been ical membrane to the subapical region after transfer to detected in the intestine of the eel [149], and the expres- SW, indicating decreased transcellular water permeabil- sion level is higher in the rectums of SW-acclimated fish ity across the intestinal epithelium [139]. Thus, it is hy- than in those of FW fish [110]. The aqp3 expression is pothesized that water is transported mostly via the low in the intestines of Mozambique tilapia [254] and paracellular route after SW acclimation in medaka, Takei Zoological Letters (2021) 7:10 Page 14 of 34

although paracellular water permeability appears to be more widely along the crypt-villus axis [98]. Accordingly, it suppressed in salmonids after SW acclimation [213]. is controversial whether the same enterocytes have both ab- The water permeability of TJ proteins is discussed in 6.1. sorptive and secretory functions or whether two different cell types exist. In the eel, guanylin has been shown to in- − 4.3 Cl− secretion hibit NKCC2 and stimulate apical Cl channels at the same − As shown in Table 1, SW contains similar concentra- time via a single second messenger, cGMP, resulting in Cl − tions of Na+ (450 mM) and Cl (524 mM). As esophageal secretion [9]. Thus, a single enterocyte seems to be able to − desalinization removes Na+ and Cl equally from SW, change its function from absorption to secretion (see 5). − Na+ and Cl concentrations in the luminal fluid may However, a small population of secretory-type enterocytes also be similar when ingested SW enters the intestine appear to exist in the killifish intestine [145]. for absorption. In the anterior intestine, NKCC2 takes Typical secretory-type cells are characterized by the − up 1Na+ and 2Cl from the luminal fluid, and AE further presence of cystic fibrosis transmembrane regulator − − − takes up Cl in exchange for HCO3 (Fig. 3). Accord- anion (Cl ) channels (CFTRs) on the apical membrane − ingly, the amount of Cl in the luminal fluid decreases and NKCC1 (SLC12a3) on the basolateral membrane in much faster than that of Na+ during passage along the mammals (Fig. 5). Low cytosolic Na+ caused by NKA − − intestinal tract. However, the concentration of Cl in the promotes the activity of NKCC1 to take up Cl from the luminal fluid is higher than the Na+ concentration in the extracellular fluid into the cell. Then, increased cytosolic − intestine and rectum in eels (Table 1) and other marine Cl is secreted into the intestinal lumen via CFTR, which − teleosts [4, 69, 182]. This implies that Cl is secreted is facilitated by the negative intracellular potential pro- into the lumen to maintain the activity of NKCC2 and duced by NKA. Anion secretion accompanies parallel AE for constant NaCl absorption. fluid secretion into the lumen, resulting in secretory − In mammals, Cl secretion has been demonstrated in diarrhea in mammals [61]. The suite of transporters for − crypt cells of the intestine (see [61]). The secretory-type Cl secretion in the intestine is similar to those of cells were once thought to be restricted to the crypt region mitochondrion-rich ionocytes of the gills in marine tele- of the intestine, but it was later shown that they are present osts [85, 86] and secretory epithelial cells of the rectal

Fig. 5 Transporters involved in transcellular Cl− secretion (blue rectangles) into the lumen by intestinal epithelial cells in mammals (A) and seawater (SW)-acclimated eels (B). Because of the low expression of NKCC1 and CFTR in the intestines of SW eels, alternative molecular mechanisms are suggested. For details, see 4.3.1. For abbreviation definitions, see the list Takei Zoological Letters (2021) 7:10 Page 15 of 34

− gland in marine elasmobranchs [203]. In this sense, the membrane for Cl absorption, as suggested in killifish rectal gland is similar to the crypt cells of the mamma- [145]. Gene expression does not always parallel protein lian colon. abundance in cells, and it is possible that CFTR protein resides on the apical membrane or is stored in vesicles 4.3.1 Possible molecular mechanisms in the subapical region in enterocytes for recruitment In the teleost intestine, Marshall et al. [146] found sub- after stimulation. In both FW and SW eel intestines, stantial expression of cftr as high as that in the gills of however, immunoreactive CFTR is not found on the ap- SW-acclimated killifish. Apical localization of immuno- ical membrane but rather in cytoplasmic vesicles [269]. − reactive CFTR occurs only in the enterocytes of SW fish Thus, it is likely that apical Cl channels different from − [145]. The enterocytes with CFTR immunoreactivity at CFTR may exist for Cl secretion in the eel. The pres- − the brush border membrane compose 20% of the total ence of DPC-inhibitable and guanylin-sensitive Cl enterocyte population, suggesting the presence of channels has been shown on the apical brush-border secretory-type cells (Fig. 5). As immunoreactive CFTR membrane of the eel intestine (see 5). ClC2 is usually lo- − migrates to the basolateral membrane in FW, the calized to the basolateral membrane for Cl absorption − function of CFTR may change from Cl excretion in SW in the intestines of mammals [28], as illustrated in Fig. 3. − − to Cl absorption in FW (see 6.4). Substantial expression However, guanylin stimulates Cl secretion in the intes- − − of cftr has also been detected in the intestines of tines of Cftr / mice, where apical ClC2 is suggested to − Mozambique tilapia [130] and spotted sea bass [282], compensate for CFTR for Cl secretion (see [56]). As the and the expression is enhanced in hypersaline environ- polarity of vesicle recruitment changes depending on the ments in these fishes. The cftr expression has also been environmental stimuli, as suggested for CFTR [145], it is − reported in the intestines of gilthead sea bream [66], possible that ClC2 is responsible for Cl secretion stimu- three-spine stickleback [140], and European sea bass [4]. lated by guanylin (unpublished data). Alternatively, it is In the sea bream, cftr expression is downregulated in the also possible that SLC26a6 on the apical membrane ex- − 2− anterior intestine after transfer from diluted 12-ppt SW cretes Cl in exchange for SO4 driven by its high con- to regular SW or to hypertonic 55-ppt SW, but it is up- centration (> 100 mM) in the luminal fluid (see 6.2) or − − regulated in the rectum. that SLC26a3 excretes Cl in exchange for HCO3 − The partner of apical CFTR for transcellular Cl secre- driven by its high concentration (~ 100 mM) in the lu- tion is basolateral NKCC1, as mentioned above. This minal fluid (see 4.4.1). SLC26a6 in the SLC26 family has 2− coupling may also be the case in the intestines of some a high affinity for SO4 [199], and it actively exchanges 2− − teleost species. The expression of two NKCC1 genes SO4 and Cl in the renal proximal tubules of eels − − (slc12a2a and slc12a2b) has been demonstrated in the [256], while SLC26a3 readily exchanges Cl and HCO3 intestine of the spotted sea bass [282]. The expression of [199]. both NKCC1 genes in the intestine is much lower than Concerning NKCC1 on the basolateral membrane, that in other tissues and much lower than that of the only slc12a2a is expressed in the intestines of both CFTR gene. Furthermore, the expression of the major European eels [37] and Japanese eels [271]. Compared isoform, slc12a2a, does not change after acclimation to a with that of the NKCC2 gene (slc12a1b), slc12a2a ex- hypersaline medium. Two NKCC1 genes are also pression is low, and it does not change after SW accli- expressed in the intestine of the gilthead sea bream [66], mation (Fig. 4). In addition, slc12a2a expression is three-spine stickleback [140], and European sea bass [4]. higher in silver eels ready for downstream migration Similar to cftr expression, slc12a2 expression is down- than in river-dwelling yellow eels [37]. Because of the regulated in the anterior intestine of the sea bream but low slc12a2 expression in the eel intestine, sulfate anion upregulated in the rectum after transfer from an isotonic transporter 1 (SAT1, SLC26a1) could be a candidate for − 2− to a hypertonic medium. Cl uptake at the serosal side in exchange for SO4 , 2− In contrast to the roles in these teleost species, the which is facilitated by uptake of SO4 from the luminal − − 2− roles of CFTR and NKCC1 in Cl secretion seem to be fluid by SLC26a6 (Fig. 5, see 6.4). Cl /SO4 exchange minor in the eel. The expression of two cftr isoforms activity has been demonstrated to occur in the basolat- (cftra and cftrb) is detectable along the intestinal tract in eral membrane of the rabbit intestine [191]. Japanese eels [269]. However, the expression of cftra,a When all the data obtained thus far are taken to- dominant isoform in the intestine, is low compared with gether, it seems that secretory-type enterocytes, which that of clcn2 and clcn7, which are upregulated in SW- have CFTR on the apical membrane and NKCC1 on acclimated eels (Fig. 4). Furthermore, the expression of the basolateral membrane, may exist in the marine − cfrta is downregulated in the anterior intestine in SW- teleost intestine for Cl secretion into the lumen (Fig. − acclimated fish (Fig. 4). It is possible that CFTR of the 5). This Cl compensation in the luminal fluid allows FW eel intestine is localized in the basolateral constant activity of NKCC2 and AE/NHE to be Takei Zoological Letters (2021) 7:10 Page 16 of 34

maintained for NaCl absorption and thus water ab- mucosal side [73, 263], and the presence of AE on sorption. This mechanism enables marine teleosts to the apical membrane is now widely recognized [68]. absorb > 80% of water from ingested SW and explains Concerning inhibition by serosal DIDS, as removal of − + − why the luminal fluid Cl concentration is higher than serosal Na profoundly decreases HCO3 secretion in + + − the Na concentration in the luminal fluid of marine the eel [7], Na -HCO3 cotransporter (NBC), a mem- − teleost intestine. However, knowledge from the eel in- ber of the DIDS-sensitive SLC4 family of HCO3 − testine suggests that an alternative set of transporters transporters [174], may be involved in HCO3 uptake − is responsible for Cl secretion, which requires further from the serosal side for secretion into the lumen. − clarification. Intestine-specific knockdown of a trans- Two sources of HCO3 in the cell are conceivable for − − porter gene can be performed evaluate the genes re- HCO3 secretion into the lumen. One is HCO3 taken sponsible for SW acclimation. The use of membrane- up from the serosal side by NBC [231] as mentioned permeable antisense oligonucleotides injected directly above, facilitated by NKA-induced low cytosolic Na+ [68, − into the intestinal lumen enables intestine-specific 69, 71]. The other is endogenous HCO3 produced in gene knockdown. the enterocyte by hydration of CO2 catalyzed by cyto- solic CAII [230]. Because of the high metabolic activity − 4.4 HCO3 secretion and carbonate precipitation of the cells, CO2 production in intestinal epithelial cells The high CO2 concentration and high pH of the rectal is high. In fact, both sources contribute to the increase − fluid in marine teleosts were recognized 90 years ago by in cytosolic HCO3 in the teleost intestine, while their Homer W. Smith [207]. It was shown later that the high relative contributions differ considerably among species − − pH is caused by active HCO3 secretion into the lumen [69]. Since serosal DIDS and removal of HCO3 in the − by epithelial cells (see [68, 69, 259]). Luminal fluid alkali- serosal fluid profoundly decrease HCO3 secretion in zation is more profound after fish are acclimated to the goby [43], eel [7] and other marine teleosts [262], − − hypertonic SW [264], suggesting a role of HCO3 secre- the contribution of serosal HCO3 is significant in these − tion in SW acclimation. As will be discussed in detail species. On the other hand, almost all HCO3 is supplied − below (see 4.4.2), the secreted HCO3 helps precipitate by CO2 hydration in SW-acclimated rainbow trout [75]. carbonates of divalent ions (Mg2+ and Ca2+), which are CA is one of the enzymes that has the fastest reaction present in SW at concentrations higher than those in rate and catalyzes the following reaction: CO2 +H2O ⇌ − + plasma (Table 1) and further concentrated by water ab- HCO3 +H . The forward reaction is much faster than sorption. Precipitate formation decreases luminal fluid the reverse reaction, and in high-CO2 environments osmolality and further enhances water absorption [73]. such as intestinal epithelial cells. Thus, the presence of − It is important to note that carbonate precipitate forma- cytosolic CAII greatly enhances HCO3 production. − tion by marine teleost intestines explains 3–15% of the Under high-HCO3 and high-pH conditions, such as in total oceanic carbon cycle and contributes to the the intestinal lumen, however, the reverse reaction oc- amelioration of ocean acidification via fixation of CO2 in curs, which is accelerated by membrane-bound CAIV the ocean [265]. Because of its important role in global (see below). − sustainability, HCO3 secretion by the marine teleost − intestine is one of the recent topics of interest in fish 4.4.1 Molecular mechanisms of HCO3 secretion − physiology [48, 67, 80, 87]. The molecular mechanisms of HCO3 secretion have − The involvement of apical AE in HCO3 secretion has been investigated for more than a decade in several tele- − been suggested by in vitro experiments using Cl -defi- ost species, and ample data have been accumulated [66, cient Ringer solution or application of DIDS on the mu- 69, 122, 231, 259]. In this section, however, we will first cosal side of the intestinal epithelium. Consistently, describe the data obtained in eels for comparison with − mucosal application of DIDS inhibits HCO3 secretion those obtained in preceding studies on other teleost in the sanddab, Citharichthys sordidus, and the rainbow species. trout [74, 75]. However, DIDS is more potent in inhibit- − ing HCO3 secretion when applied to the serosal side Mucosal side Three SLC26a6 genes (slc26a6a, slc26a6b, than to the mucosal side; it is only slightly inhibitory slc26a6c) are expressed at significant levels in the eel in- when applied to the mucosal side in the eel [7] and other testine, as assessed by transcriptomic analyses followed teleost species [43, 52]. Such species differences may be by real-time qPCR for differentiation of the isoforms due to the differences in major transporters used (Figs. 6 and 7A). Among the isoforms, slc6a6a exhibits among species [224] and the different sensitivities of the most abundant transcripts in the posterior intestine, AEs to DIDS among teleost species, as shown in and the transcript levels decrease in the anterior direc- − mammals (see below). HCO3 secretion is significantly tion; in contrast, slc26a1b transcripts are most abundant − inhibited when Cl -deficient Ringer solution is on the in the anterior intestine, and the transcript levels Takei Zoological Letters (2021) 7:10 Page 17 of 34

− Fig. 6 Major transporters responsible for HCO3 secretion (red rectangles) into the lumen by the intestinal epithelial cells of SW eels. These transporters also function in NaCl absorption (see Fig. 3) and Mg/CaCO3 precipitation (see Fig. 8). NHEs, SLC26a3/6 s and CFTRs are combined as a metabolon for mutual activity regulation (see 6.3 and Fig. 8). The text size for the transporters is related to the relative abundance and upregulation of the transporters in SW. For details, see 4.4.1. For abbreviation definitions, see the list

decrease in the posterior direction. The slc26a6c expres- slc26a6a/b (Fig. 7A). The stoichiometry of SLC26a3 is − − sion is the lowest among these isoforms in all intestinal reported to be HCO3 /2Cl in mammals (see [106]), but segments. The slc6a6a expression is upregulated in all it has not yet been examined in teleosts. As mentioned − segments of the intestine during the course of SW accli- above, however, mucosal DIDS fails to decrease HCO3 mation [228], although the expression returns to the FW secretion in the eel intestine [7, 228]. Although DIDS is level after SW acclimation (Fig. 7A). The stoichiometry generally an effective inhibitor of AE of the SLC26 fam- of SLC26a6 has been suggested to be electrogenic, ex- ily in mammals, it fails to block SLC26a3 in some spe- − − changing 2HCO3 with 1Cl , in the mefugu [107], while cies [15, 260], showing species specificity of DIDS for variable stoichiometry has been reported in mammals [1, SLC26a3. Among teleosts, DIDS effectively blocks 106]. The electrogenic nature of SLC26a6 has also been SLC26a6 in the rainbow trout [22], but its effect on shown in mefugu [122] and toadfish [76]. SLC26a3 has not been confirmed in any teleost species. In addition, two SLC26a3 genes (slc26a3a and Supposing that DIDS is effective for SLC26a6 but inef- slc26a3b) are expressed abundantly in the eel intestine, fective for SLC26a3 in eels, the major apical AE of the − − and their expression is upregulated profoundly after SW SLC26 family responsible for HCO3 secretion and Cl transfer until the eels are fully acclimated to SW (Fig. 6 absorption might be SLC26a3 in eels. − and 7A). The slc26a3b expression is most abundant in Other candidate transporters for HCO3 secretion are the anterior intestine and decreases in the posterior dir- anion channels on the apical membrane, and cftra ex- ection, while slc26a3a expression is most abundant in pression has been detected in the eel intestine (Fig. 4). − the posterior intestine and decreases in the anterior dir- CFTR is known to readily pass HCO3 in mammals − ection. In this way, two SLC26a3 isoforms and two [204] and couple with SLC26a3/6 to stimulate HCO3 SLC26a6 isoforms appear to compensate for each other secretion [212] (Fig. 8). The polarity (apical or basolat- in different segments of the intestine. The expression eral) of CFTR localization seems to be variable depend- level of slc26a3a/b is several-fold higher than that of ing on the environmental salinity and other conditions Takei Zoological Letters (2021) 7:10 Page 18 of 34

A. SLC26 family AEs slc26a3a slc26a3b slc26a6a 0.10 0.3 0.015 ** 1a) 0.08 ef 0.2 0.010 0.06

0.04 * ve to 0.1 0.005 i t 0.02 * * 0.00 0.0 0.000 AI MI PI AI MI PI AI MI PI n(rela o i

s slc26a6b slc26a6c slc26a1 0.08 0.0015 0.015

xpres 0.06 0.0010 0.010 ee 0.04 en

G 0.0005 0.005 0.02 * 0.00 0.0000 0.000 AI MI PI AI MI PI AI MI PI B. SLC9 family NHEs slc9a1 slc9a2 slc9a3

0.0015 0.005 0.010 on i 0.004 0.008 0.0010 0.003 0.006 press x

e 0.002 0.004 0.0005 e 0.001 0.002

Gen 0.0000 0.000 0.000 AI MI PI AI MI PI AI MI PI C. SLC4 family AEs slc4a1a slc4a2a slc4a2b

n 0.00025 0.003 0.010 io 0.00020 0.008 0.002

ress 0.00015 0.006

p * 0.00010 0.004 ex 0.001

e 0.00005 0.002

0.00000 0.000 0.000 Gen AI MI PI AI MI PI AI MI PI D. SLC4 family NBCs

slc4a4 slc4a7 slc4a10 0.6 0.008 0.0025 *** 0.0020 0.006 ** 0.4 0.0015 * *** 0.004 0.0010 0.2 0.002 0.0005

Gene expression 0.0 0.000 0.0000 AI MI PI AI MI PI AI MI PI

E. Carbonic anhydrases ca2a ca2b ca4

n 0.004 0.04 0.15

ssio 0.003 0.03 ** * 0.10 0.002 0.02 * 0.05 0.001 0.01

Gene expre 0.000 0.00 0.00 AI MI PI AI MI PI AI MI PI

F. V-ATPases atp6v1a atp6v1b1 atp6v1b2 0.06 0.015 0.0015 * *** 0.04 0.010 0.0010

* 0.02 0.005 0.0005

Gene expression 0.00 0.000 0.0000 AI MI PI AI MI PI AI MI PI Fig. 7 (See legend on next page.) Takei Zoological Letters (2021) 7:10 Page 19 of 34

(See figure on previous page.) − Fig. 7 Expression of the genes responsible for HCO3 secretion in the anterior (AI), middle (MI) and posterior (PI) intestine in FW-acclimated (plain column) and SW-acclimated (filled column) eels as determined by real-time qPCR. The genes are grouped by SLC family and function. The atp6sare the genes for of VHA subunits. The figures are based on the data in Takei et al. [228] and Wong et al. [269] and on unpublished data. The primers for real-time qPCR, including those for the unpublished data, are listed in Supplementary Table 1.*p <0.05,**p < 0.01, ***p < 0.001. For details, see 4.4.1. For abbreviation definitions, see the list

[145]. clcn2 and clcn3 are expressed significantly in the found in sea bream [66] and tilapia [183] after hyperos- eel intestine (unpublished data). Significant expression motic stimulation. The slc26a3 and slc26a6 expression of clcn3 has also been reported in the cod intestine [87]. has also been detected in the intestine of the European However, the major role of apical ClCs appears to be for sea bass [4]. It seems that the relative contributions of − − Cl secretion in teleost fishes (see 4.3.1). SLC26a3 and SLC26a6 to HCO3 secretion differ among The involvement of SLC26a6 was first suggested in the teleost species. euryhaline mefugu, in which slc26a6 expression is pro- As partners of SLC26a3 and SLC26a6 for coupled up- + − + − foundly upregulated after transfer from FW to SW take of Na and Cl and excretion of H and HCO3 , [122]. Similar results have been reported in the rectum three NHE genes, NHE1 (slc9a1), NHE2 (slc9a2) and in gulf toadfish [179] and gilthead sea bream [66] trans- NHE3 (slc9a3), are expressed in the eel intestine (Fig. 6 ferred from diluted SW to concentrated SW. In contrast, and 7B). NHE1 and NHE2 are resident-type, while slc26a6 expression in the anterior intestine of NHE3 is a mobile-type transporter that is stored in cyto- Mozambique tilapia is suppressed after transfer from plasmic vesicles and inserted into the plasma membrane FW to SW [183]. In addition, slc26a3 expression has (see 6.4). The expression of NHE genes does not change also been detected in the intestines of several teleost in the eel intestine after SW transfer (Fig. 7B). AE and species, and upregulation of the transcripts has been NHE are thought to be physically associated in the

+ − Fig. 8 A suite of transporters and enzymes form a metabolon at the apical membrane of enterocytes for H and HCO3 secretion and recycling to form carbonate precipitates in SW eels. VHA may also be involved in H+ secretion into the lumen (see Fig. 7). This idea of metabolon formation was + − first proposed in the mammalian intestine and may be applicable to the teleost intestine (see 6.3). H and HCO3 secreted into the lumen by apical transporters are catalyzed by membrane-bound CAIV to produce CO2 and H2O, which are recycled into the cell via AQP1. Cytosolic CAII then produces + − H and HCO3 again for continuous supply of the ions into the lumen. NHE3, SLC26a3/6 and CFTR are bound on NHERF for mutual regulation. CAIV also facilitates Mg/CaCO3 production in the microenvironment under the mucus layer, which is maintained by continuous secretion of mucus from goblet cells (see 4.5). CAII is known to bind to the NHE/AE complex and to AQP to regulate their activity. CFTR and SLC26a3/6 bind through the R domain and the STAS domain of each transporter. For details, see 6.3. For abbreviation definitions, see the list Takei Zoological Letters (2021) 7:10 Page 20 of 34

anchor protein (see 6.3). The gene of the anchor protein the results obtained thus far, it seems that transcellular − for NHE3, NHERF1 (slc9a3r1), is expressed significantly HCO3 transport occurs principally via apical SLC26a3 in the intestine, and its expression is upregulated two- and SLC26a6 and basolateral NBCe1 and SLC26a1/AE2 fold after SW acclimation, as demonstrated by RNA-seq in marine teleosts (Fig. 6). (unpublished data). NHE3 and AE in the anchor protein are recruited from the cytoplasmic vesicles to the apical Carbonic anhydrases and proton ATPase In addition − membrane upon phosphorylation (see 6.4). NHE3 is an to uptake from the serosal side, cytosolic HCO3 is pro- + + electroneutral exchanger of 1Na /1H . With regard to vided by hydration of CO2 in epithelial cells, which is other teleosts, nhe3 expression has been detected in the catalyzed by cytosolic CAII (CAc), as suggested in sev- intestines of rainbow trout [72] and cod [87]. eral teleost species [66, 75, 187]. CO2 is produced by ac- tive metabolism of enterocytes and is taken up from the Serosal side On the basolateral membrane, SLC26a1 luminal fluid (Fig. 8). Two cytosolic CAII genes (ca2a − may be a major AE for HCO3 uptake into the cell, as and ca2b) and a membrane-bound CAIV gene (ca4a) its gene expression is consistently upregulated in all seg- are expressed at high levels in the eel intestine, and ca2b ments of the eel intestine after SW transfer [228], al- and ca4a expression is upregulated after SW transfer though the upregulation is not significant in the middle (Fig. 7E). CAIV has a catalytic site on the luminal side + − and posterior intestine after full acclimation to SW (Fig. and may produce H2O and CO2 from H and HCO3 2− 7A). As SLC26a1 exhibits high affinity for SO4 ,itis secreted into the microenvironment between the mucus possible that apical SLC26a6 and basolateral SLC26a1 layers and the apical membranes of epithelial cells (Fig. − work in concert for transcellular secretion of HCO3 in 8), as observed in rainbow trout [72]. H2O and CO2 pro- 2− exchange for SO4 absorption in the SW eel intestine duction decrease luminal fluid osmolality to facilitate (see 6.2). Cooperation of the two sulfate transporters has water absorption, and the generated CO2 is recycled into been observed in the proximal tubules of SW eel kidneys the cell for hydration by CAII (see 4.4.2). It is likely that [257]. The SLC4 family of AEs also contains candidates CO2 is recycled efficiently via AQP1 on the apical mem- − for HCO3 secretion: two AE2 genes (slc4a2a and brane, as suggested in zebrafish [31] and mammals − slc4a2b) are expressed in the eel intestine, and slc4a2a [171]. As HCO3 secretion is inhibited to different de- tends to be upregulated after SW transfer (Fig. 7C). AE2 grees by mucosal application of acetazolamide, which may be localized on the basolateral membrane, as as- blocks both CAII and CAIV at the same time, the rela- − sumed from its location in the intestines of mammals tive contributions of the CAs to HCO3 secretion differ − [174] and by the lack of inhibition of HCO3 secretion among teleost species [69, 259]. The role of CAII in − by apical application of DIDS in the eel intestinal epithe- HCO3 secretion has been documented in the rainbow lium [228]. trout [72] and other marine teleosts [69]. − − Among other HCO3 transporters, three NBC genes, HCO3 secretion is mediated by mutual activation of NBCe1a (slc4a4a), NBCn1 (slc4a7) and NBCn2 SLC26-family AEs and CFTR on the apical membrane − − (slc4a10a), are expressed in the SW eel intestine, of (see 6.3). As SLC26a6 exchanges 1Cl /n HCO3 in tele- which slc4a4a is expressed most profoundly (Fig. 7D). osts [107], additional H+ secretion into the lumen neu- − All the NBC genes are upregulated in different segments tralizes the secreted HCO3 to support the activity of of the eel intestine after SW transfer [228]. Thus, coupled SLC26a6 and NHE3, which exchanges 1Na+/ − + + + − HCO3 may be taken up with Na from the extracellular 1H [69, 76]. In fact, H secretion facilitates HCO3 se- fluid by electrogenic NBCela and electroneutral NBCn1 cretion and increases the production of CO2 and H2O and NBCn2a. The stoichiometry of NBCe1 is 1Na+/ by CAIV (see below). H+ secretion also helps protect − + 2HCO3 in mammals [174] and electrogenic in toadfish against cellular acidification [76]. The candidate H [231]. However, the external Na+ concentration affects transporter is VHA, which is expressed in the intestines the stoichiometry of mefugu NBCe1, and the ratio be- of various teleost species, such as rainbow trout [72], + − + comes 1Na /4HCO3 at 120 mM Na , which is similar toadfish [76], sea bream [66], cod [87], tilapia [183], to the concentration in the extracellular fluid of teleosts Senegalese sole [182], and sea bass [4]. VHA is a large [29]. NBCe1 activity is facilitated by the low levels of protein complex consisting of two domains, V0 and V1, cytosolic Na+ produced by NKAs on the basolateral that consist of six and eight subunits, respectively. Sig- membranes of enterocytes [69, 133]. The slc4a4 is also nificant expression of the A subunit and 2B subunits in expressed in the intestines of mefugu [122], toadfish the V1 domain (atp6v1a, atp6v1b1, atp6v1b2) has been [231], and sea bream [66], and its expression is upregu- detected in the eel intestine. Among these subunits, lated after transfer to hyperosmotic environments. The atp6v1a and atp6v1b1 are highly expressed, and their − inhibition of HCO3 secretion by serosal DIDS coincides expression is upregulated after SW transfer (Fig. 7F). with the DIDS sensitivity of NBCs [7, 43, 69]. Given all VHA also appears to be localized on the basolateral Takei Zoological Letters (2021) 7:10 Page 21 of 34

membrane to help maintain the resting potential and As mentioned above, the space between the mucus neutral pH of the cell [69]. The CAs and VHA appear to layer and the apical membrane of enterocytes is a be physically coupled to the AE/NHE complex to form a microenvironment different from the central lumen, metabolon (see 6.3). which is maintained by constant secretion of mucus + − by goblet cells (Fig. 8). H and HCO3 are secreted 4.4.2 Mechanisms of carbonate production into the microenvironment, where not only CO2 and Formation of white precipitates consisting of Ca/Mg H2O production but also carbonate production are carbonate within the intestinal lumen has been re- efficiently facilitated by membrane-bound CAIV via 2+ 2+ − ported in various marine teleosts for more than 50 the following reaction: Ca /Mg +2HCO3 ⇌ Ca/ years; for example, such precipitates have been ob- MgCO3 +H2O+CO2 [89]. This reaction not only served in southern flounder [81], rainbow trout [201], removes divalent ions and produces H2O to decrease eels [88, 153, 207], toadfish [253], European flounder osmolality but also supplies CO2 through AQP1 for − [262]andmefugu[122]. The solubilities of MgCO3 hydration to produce HCO3 via cytosolic CAII for and CaCO3 in water are 1.65 and 0.13 mmol/l at constant operation of apical AE (Fig. 8). 25 °C, respectively, while luminal fluid Mg2+ and Ca2+ concentrations are 188 and 14 mmol/l, respectively, in 5. Hormonal control the eel intestine (Table 1). Although solubility The effects of hormones are governed by the presence changes greatly according to pH and the presence of of receptors in the tissue. Thus, it is important to − other ions, concentrated HCO3 in the luminal fluid examine the presence of hormone receptors in the certainly promotes the formation of carbonates [70, digestive tract using a sensitive detection method 265]. The chemical compositions of carbonate precipi- such as RNA-seq. It is known that the gut is one of tates have been examined in teleost intestines. The the organs that produces the greatest variety of carbonate precipitate consists of Ca carbonate and/or hormones, and it synthesizes unique hormones such calcite in the flounder [81] and eel [88], and the cal- as guanylin in addition to those produced by other cite contains one-third (by weight) Mg and a small tissues such as brain-gut peptides [151, 222]. Consid- amount of Mn, producing a substance called calcian ering the data obtained thus far, it is apparent that kutnohorite, in the gulf toadfish [253]. On the other hormones that increase cAMP or cGMP as a second hand, the carbonate precipitate contains 22.6 mol% messenger inhibit NaCl and water absorption [8]. Mg, 76.9 mol% Ca and small amounts of P and S and is called magnesian calcite (Mg calcite) in the eel 5.1 Hormones that inhibit NaCl transport [153]. Chemical and structural analyses have been The most potent hormone that inhibits NaCl absorption performed on the crystalline products in the intestinal is the cardiac hormone atrial natriuretic peptide (ANP), lumens of various tropical fishes and have revealed which almost nullifies short-circuit current (Isc) in the that Mg calcite contains 0.5 to more than 40 mol% isolated intestinal epithelium of the eel when it is applied MgCO3 [184]. The precipitate of the sea bream intes- to the serosal side [10] and in the intestine of winter tine contains 54 mol% Mg2+,andMg2+ may be re- flounder [163]. The minimum effective dose of homolo- − sponsible for stabilizing the unstable mineral crystals gous eel ANP for inhibition of Isc is as low as 10 11 M [54]. The mechanisms of Mg calcite formation have in vitro, which is much lower than those of other inhibi- been investigated in marine organisms, and high-Mg tory hormones examined thus far [11] and much lower calcite that contains > 4 mol% MgCO3 is naturally than the reported effective doses of ANP in the mamma- formed by calcite-coated such as foraminifera, lian intestine [219]. The natriuretic peptide (NP) family echinoids and corals [131]. The ratio of MgCO3 in- is highly diversified in teleost fishes, and at least 7 mem- creases in proportion to the relative Mg2+ concentra- bers, ANP, B-type NP (BNP), ventricular NP (VNP), and tion in the medium. Recently, a protein-based matrix 4 types of C-type NPs (CNP1 ~ 4), exist in the eel [93, with strong Ca2+-binding properties was found in the 94]. Biological NP receptors (GC-A and GC-B) are teleost intestine; this matrix facilitates carbonate pre- single-stranded receptors with a cytosolic guanylyl cy- cipitation [188].Theproteinswerepurifiedfromthe clase (GC) domain that produces cGMP as an intracellu- Ca precipitates in the toadfish intestine and analyzed lar second messenger consequent to ligand binding [14]. by proteomics. Many matrix proteins were found to Similar to the ligands, NP receptors are diversified in be highly acidic, which may provide negatively fishes compared with mammals [222]. The increased charged domains for immobilization of Ca2+. cGMP after ligand binding activates protein kinase GII Immobilization increases the local Ca2+ concentration (PKGII), which inhibits NKCC2 and decreases Isc by − in the microenvironment and facilitates the precipita- inhibiting K+ efflux on the mucosal side and Cl efflux − tion of divalent ions with secreted HCO3 . on the serosal side (see Fig. 3), resulting in inhibition of Takei Zoological Letters (2021) 7:10 Page 22 of 34

NaCl absorption [10, 163]. As ANP and VNP are synthe- players in Na+ absorption in mammals (see 4.1.1). In sized locally in the intestine, these hormones may act in toadfish, where AE and NHE play important roles in a paracrine fashion as well as in an endocrine NaCl absorption, renoguanylin inhibits AE (SLC26a6) to − − fashion [137]. decrease Cl absorption and HCO3 secretion [178]. In − Other hormones that potently inhibit NaCl absorption contrast, guanylin stimulates HCO3 secretion in the are the guanylin-family peptides, which are highly SW eel intestine, not through direct action on apical AE expressed locally in the intestine. The family members but through inhibition of NKCC2 [228]. NKCC2 inhib- − (guanylin, uroguanylin and renoguanylin) and their re- ition decreases cytosolic Na+ and Cl , which stimulate ceptors (GC-C1 and GC-C2) are more diversified in the apical AE and/or basolateral NBC, resulting in increased − − eel [34, 35, 278, 279] than in mammals, in which they HCO3 secretion. Thus, guanylin supplies Cl to the − have two ligands and one receptor. The guanylin family lumen for continuous NKCC2 activity, increases HCO3 and its receptors form the only known hormonal system secretion for carbonate precipitation, and flushes out whose genes are all upregulated after transfer to SW. carbonate precipitates from the rectum [12, 177], all of GC-C is another type of single-stranded membrane re- which facilitate SW acclimation, although inhibition of ceptor with intracellular GC domain in addition to the NKCC2 is certainly disadvantageous for SW acclimation. NP receptors GC-A and GC-B. Homologous eel guany- As reported recently, transfer of rainbow trout to SW lin profoundly decreases Isc or even reverses it when ap- stimulates intestinal motility [24]. As guanylin is upregu- plied to the mucosal side of the eel intestine [12, 277]. lated after SW transfer in the eel and toadfish intestine The Isc inhibition is due to inhibition of NKCC2, as is and activates intestinal motility in mammals [55], the in- the case for ANP. Similar effects have been observed in creased motility could be due to guanylin. the toadfish using eel renoguanylin, a heterologous hor- It has been suggested that guanylin-induced Isc inhib- mone that is similar to toadfish guanylin [177]. The ition is mediated not by cGMP but by cAMP in mam- − minimum effective dose for inhibition is 10 9 M[12], mals [14]. This is because the cGMP increased by which is higher than that for ANP [11]. This may be due guanylin inhibits cAMP-specific phosphodiesterase to the paracrine action of guanylin from the lumen, into 3 (PDE3), resulting in increased cAMP levels in entero- which it is secretedwith mucus from goblet cells and cytes. Transcriptome analysis has shown that the PDE3 where it acts on GC-C localized on the apical mem- gene is also expressed in the eel intestine (unpublished branes of enterocytes (acting in a luminocrine manner) data). Notably, the effect of renoguanylin on intestinal − − [278]. Guanylin is more effective on the mucosal side Cl and HCO3 secretion is suggested to be mediated by than on the serosal side of the intestinal epithelium PKA in the toadfish [180]. Thus, hormones that increase in vitro. Thus, guanylin is a local hormone in the intes- cytosolic cAMP inhibit Isc and therefore NaCl/water ab- tine, although it is also secreted into the circulation. sorption in teleost fishes. This indicates that hormones Guanylin and uroguanylin secreted into the circulation that bind to G protein-coupled receptors (GPCRs) asso- may act directly on the kidneys to inhibit NKCC2 at the ciated with the Gαs subunit are inhibitory to transport. TAL and induce diuresis/natriuresis [56]. Such hormones include vasoactive intestinal peptide, More recent studies have shown that guanylin not only whose inhibitory action has been demonstrated in the − inhibits NKCC2 but also stimulates Cl channels on the intestines of winter flounder [162], Mozambique tilapia − apical membrane for Cl secretion into the lumen in the [143] and eels [11]. Urotensin I is also inhibitory in the eel [9] and toadfish [177], resulting in reversal of Isc.In goby [136], in which it binds to Gαs-associated GPCRs. the eel, this effect is only detectable when fish Ringer so- On the other hand, stimulation of soluble adenylyl cy- lution on the mucosal side is replaced with a simulated clase (sAC) is suggested to be stimulatory for Isc in the luminal fluid in vivo that contains low NaCl and high toadfish intestine [237]. It has been shown that increased − MgSO4 and when NKCC2 activity is inhibited. The en- HCO3 concentrations in Ringer solution on both sides − hanced Cl secretion by guanylin occurs via DPC- of an Ussing chamber increase Isc and that this increase − − sensitive Cl channels, not CFTR, in the apical mem- disappears following removal of mucosal Cl and muco- brane. In the toadfish, on the other hand, guanylin- sal application of bumetanide, suggesting an involvement − induced Cl secretion appears to be mediated by apical of NKCC2. The findings also suggest that NKCC2 acti- CFTR and basolateral NKCC1, as in mammals [177, vation is induced by sAC because sAC inhibition abol- − 180]. In the mammalian intestine, where NKCC2 is ishes the stimulatory effect of HCO3 on Isc [237]. This scarcely present on the apical membrane, guanylin in- idea is interesting and suggests a new role for sAC in − − duces Cl and HCO3 secretion into the lumen through the regulation of intestinal function. However, all hor- activation of apical CFTR [14, 56]. Guanylin inhibits Na+ mones that increase cytosolic cAMP inhibit Isc. Calvalho absorption in mammals through inhibition of NHE3 and et al. investigated this issue and found that transmem- the scaffolding protein NHERF2, which are the major brane AC activated by hormones and sAC exert opposite Takei Zoological Letters (2021) 7:10 Page 23 of 34

effects on Isc in the sea bream intestine, although both 197, 249]. Cortisol implantation induces dose-dependent ACs produce the same effector, cAMP, in the cytoplasm increases in NKA activity in the posterior intestine and [26]. As mentioned above, NKCC2 governs the whole pyloric caeca of chinook salmon [248]. However, the ef- transport system in the intestines of most marine tele- fect of cortisol on water transport is slow, suggesting osts, and inhibition of NKCC2 profoundly affects other that transcriptional activation of transporters occurs transporters and electrical parameters involved in intes- [247]. − tinal transport, as exemplified by guanylin-induced Cl − − secretion [9] and HCO3 secretion [228]. More data may 5.3 Hormones that affect HCO3 transport − be required to define the stimulatory effect of sAC on The effects of calciotropic hormones on HCO3 secre- NKCC2, including data on the specificity of sAC inhibi- tion have been investigated in the teleost intestine in re- tors in teleosts. lation to CaCO3 precipitation. In teleosts, parathyroid hormone-related peptide (PTHrP) and stanniocalcin 1 5.2 Hormones that stimulate NaCl transport (STC1) are the major hypercalcemic and hypocalcemic In contrast to inhibitory hormones, hormones that de- hormones, respectively, while calcitonin seems to have crease cAMP appear to increase Isc and NaCl absorption minor roles in Ca2+ metabolism in teleost fishes. Serosal in the teleost intestine. Examples are somatostatin [245] application of pufferfish PTHrP increases 45Ca2+ influx and neuropeptide Y [246] in eels, whose receptors are in vitro in the seabream intestine [58], whereas salmon GPCRs associated with the Gαi/o subunit that decrease STC1 perfusion into the isolated cod intestine decreases cytosolic cAMP after ligand binding. Urotensin II and Ca2+ influx [214]. In intestinal epithelia mounted in − AVT also increase Isc in the intestines of tilapia [143] Ussing chambers, PTHrP decreases HCO3 secretion, − and sea bream [150]. These hormones bind to GPCRs while STC1 increases HCO3 secretion in the sea bream associated with the Gαq/11 subunit and increase the [59]. Interestingly, PTHrP decreases water absorption in 2+ levels of the second messengers IP3/Ca and diacylglyc- intestinal sacs of sea bream [27]. As the in vitro experi- erol (DAG). As the seabream intestine used in the above ments revealing these findings used the same fish Ringer experiment shows low serosa-negative or serosa-positive solution on both sides of the intestinal epithelium, it PD, unlike the high serosa-negative PD in other marine would be intriguing to examine the effects of PTHrP teleosts [133], NKCC2 may be inhibited before AVT using simulated in vivo luminal fluid on the mucosal stimulation in the seabream intestine. For instance, the side, in which carbonate precipitation could occur. As − Isc of the intestine in SW-acclimated eels is ~ 500 μA/ mentioned above, renoguanylin inhibits HCO3 secre- cm2 [12], while it is less than 5 μA/cm2 in the seabream tion in the toadfish intestine [180], while guanylin − intestine [150]. AVT does not affect Isc in the intestines stimulates HCO3 secretion in the eel intestine of tilapia [142] or eels (unpublished data), which exhibit when simulated luminal fluid was used on the mucosal − high serosa-negative PD caused by high NKCC2 activity side [228]. In mammals, guanylin stimulates HCO3 se- (Fig. 3). It is possible that AVT-induced increases in IP3/ cretion from the duodenal epithelium in vitro [77] and DAG levels release the inhibition of NKCC2 in the seab- in vivo [17] through activation of CFTR. Prolactin de- − ream intestine. The different effects of AVT also exem- creases HCO3 secretion in the seabream intestine plify the diversity of osmoregulatory mechanisms among in vitro [53]. Interestingly, incubation of anterior in- teleost species [224]. testinal explants with prolactin for 3 h downregulates The renin-angiotensin system is activated in the SW slc4a4 expression but not slc26a6 or slc26a3 expres- environment in teleosts [267], as indicated by elevated sion. Expression of the two prolactin receptor genes circulating levels of angiotensin II (Ang II) in the eel (prlr1 and prlr2) has been reported in the [266]. Ang II appears to inhibit NKA activity in the in- Mozambique tilapia intestine, and prlr2 expression is testine of the eel to reduce NaCl absorption [148], upregulated in SW-acclimated fish [198]. A recent re- and causes a reduction in plasma Na+ concentration in view has summarized the osmoregulatory action of concert with an Ang II-dependent increase in NKA ac- prolactin in the gastrointestinal tracts of fishes [23]. tivity in the gills and kidneys in SW eels. Ang II also in- Research on hormonal regulation of ion and water − creases water and Cl absorption in intestinal sac of transport in the teleost intestine has focused mostly on FW-acclimated tilapia but not in those of SW- the transcellular pathway, but paracellular transport acclimated fish [142]. It is possible that Ang II, whose through TJ proteins may also be regulated by hormones receptor (AT1) is a Gαq/11-coupled GPCR, releases the (see 6.1). ANP has been suggested to affect paracellular inhibitory effect of NKCC2 on NaCl absorption, as dis- ion transport in the eel intestine [239]. In addition, cussed above for AVT. Cortisol is known to enhance several hormones have been shown to affect cldn NKA activity and ion/water absorption in the intestines expression in the Atlantic salmon [235]. Among those of eels, salmonids and other model fish species [84, 151, hormones, cortisol, growth hormone and prolactin Takei Zoological Letters (2021) 7:10 Page 24 of 34

downregulate the expression of cldn15, the major CLDN function of TJs [78, 119]. TJs are not simple barriers for in the teleost intestine (see 6.1), in the anterior intestine ions and water; rather, they exhibit selectivity for the in FW salmon. This downregulation probably regulates substances that pass through the junctions according to the changes in paracellular permeability by reorganizing their concentration gradients, and CLDNs are the major intestinal tissues in preparation for downstream migra- components involved in selectivity [60, 121]. The CLDN tion. There have been several studies on the hormonal family is composed of 27 members, which are divided regulation of CLDNs in the gills [116, 117]. In addition largely into two groups: barrier-forming CLDNs, such as to the intestine, hormonal regulation of esophageal desa- CLDN1, CLDN3, CLDN4, CLDN5, CLDN6, CLDN8, linization is also likely as mentioned above (see 2.1). CLDN12 and CLDN18, and channel-forming CLDNs, such as CLDN2, CLDN10, CLDN15, CLDN16, CLDN17 6. Future directions and CLDN19. The channel-forming CLDNs are further There are a number of important questions that have subdivided into cation-selective channel formers, such as not yet been answered about the role of the intestine in CLDN2, CLDN10b, CLDN15, CLDN16 and CLDN19, SW acclimation in teleost fishes. Regarding transporters, and anion-selective channel formers, such as CLDN10a major transporters essential for NaCl and water absorp- and CLDN17 [121]. CLDN10a and CLDN10b are splice tion may have been identified using contemporary mo- variants from the same gene. Among channel-forming lecular techniques, and more will be identified in the CLDNs, only CLDN2 serves as a paracellular water near future using newly developed bioinformatics tools channel [175]. In leaky epithelia such as those of the in- that enable detection of trace expression of genes that testine and renal proximal tubules, paracellular fluid are important for fine-tuning of physiological responses. transport plays a significant role in water absorption. In A more important goal is to confirm the functions of the anterior intestines of rodents, CLDN2 and CLDN15 the identified genes, probably using genetically modified are abundantly expressed and serve as routes for Na+ re- fishes. As discussed above, the molecular mechanisms of cruitment between epithelial cells for continuous activity transport are considerably different among species, not of Na+-coupled nutrient absorption [229]. CLDN15 is only between euryhaline and stenohaline species but also the most studied CLDN, and its gene knockout results within euryhaline species such as catadromous eels and in an extraordinarily large intestine in terms of length anadromous salmonids [224]. In this regard, it is import- and diameter (megaintestine). Recently, CLDN15 has ant to thoroughly establish the molecular mechanism in been suggested to be a paracellular water channel in the one species for comparison with the mechanisms in small intestine in mammals [176]. other species. The molecules include not only The number of CLDN genes increased by more than transporters but also TJ proteins, the latter of which double in teleost fishes via teleost-specific whole- constitutes the first topic of this section. As a model genome duplication and tandem duplication of genes species, the euryhaline medaka (genus Oryzias) may be [117]. Duplicated genes are usually nonfunctionalized as advantageous because a reliable genome database is pub- pseudogenes or subjected to posttranscriptional gene licly available [104], techniques for gene modification silencing, but most of the CLDN genes are retained in have been established [112], and kin species with differ- teleosts. This retention may be related to aquatic life be- ent salinity tolerances exist [92]. The second topic of this cause the body surfaces of teleosts are exposed directly 2+ 2− section is Mg and SO4 transport in the intestine. to environmental water that dissolves various substances The intestinal epithelium has long been thought to be and has an osmolality different from that of body fluids. impermeable to these divalent ions, but they seem to be Thus, the epithelial cells of the body surface must cope significantly able to pass through the epithelium, as indi- with these issues via TJs. As a result, the gills have been cated in eel studies [257]. The third and fourth topics the major targets of CLDN research in fishes [30]. For have scarcely been investigated in fishes, so these topics instance, cation selectivity and salinity-dependent ex- will be discussed based on data obtained in mammals to pression has been examined for duplicated channel- frame future studies in fishes. forming CLDN10s in killifish gills [147]. The expression levels of cldn1, cldn5b, cldn7a, 6.1 Paracellular route cldn11a, cldn11b, cldn12, cldn15, and cldn23a have been The sheet-like polarized epithelial cells that transport detected by RNA-seq in the eel intestine, and cldn7a ions and water are joined by TJs near the apical part of and cldn15 are upregulated after SW transfer (unpub- the lateral membrane, which serve as barriers for free lished data). Real-time qPCR has not yet been performed movement of ions and water between cells [243]. Junc- on cldns expressed in the intestine. In the esophagus, tional proteins consist of several molecules, of which similar sets of cldns are expressed, and the number of CLDNs are major constituents and play essential roles in expressed genes is much greater in the esophagus than the sealing mechanism in terms of the structure and in the intestine, illustrating the watertight nature of the Takei Zoological Letters (2021) 7:10 Page 25 of 34

esophagus (see 2.1). Among the genes, cldn15 is because these ions are secreted into the urine at high expressed at the highest level, and its expression is up- concentrations in marine teleosts [18, 173, 257], al- regulated after SW transfer in the eel intestine, as in the though the fraction of renal excretion of absorbed ions esophagus [227]. Of the CLDNs expressed in the eel appears to be small in the toadfish [69]. The Mg2+ trans- esophagus, only CLDN15 is a cation channel type; the porters SLC41a1 and Cyclin M3 (Cnnm3) have been others are barrier-forming types. Because of the upregu- identified in the renal proximal tubules of SW- lation of cldn15 after SW acclimation, paracellular Na+ acclimated mefugu, and they have been suggested to be influx through TJs could be accelerated in the esophagus involved in Mg2+ secretion into the lumen [96, 97]. after drinking of SW. In the intestine, by contrast, as the Mg2+ seems to be concentrated in cytoplasmic vesicles Na+ concentration of luminal fluid is much lower than and secreted into the lumen via exocytotic delivery of + 2− that of extracellular fluid (Table 1), absorbed Na is vesicle contents [18]. The SO4 transporters SLC26a6 recirculated back into the luminal fluid via CLDN15 to (apical) and SLC26a1 (basolateral) exist in renal prox- maintain NKCC2/NCC activity. Recently, CLDN15 has imal tubule cells of SW eels [256] and SW mefugu [107], 2− been suggested to be water-permeable in mammals, but and these AEs secrete SO4 transcellularly from the ser- − Na+ influx blocks water movement through the TJ pro- osa to the mucosa in exchange for Cl . As a result, Mg2+ 2− tein [176]. Thus, even if teleost CLDN15 is also water- and SO4 concentrations in the urine of SW-acclimated permeable, water efflux may be blocked by vigorous Na+ eels are 116.1 mM and 36.5 mM, respectively, higher 2− transport caused by luminal SW in the SW eel esopha- than the concentrations in SW. In contrast, SO4 is ac- + − − gus. Although Na may move through TJs, Cl may not tively absorbed in exchange for HCO3 in the proximal tu- move in parallel through the paracellular route because bules of FW eels, where apical SLC13a1 and basolateral 2− − no candidates for anion-specific CLDNs are expressed in SLC26a1 transport SO4 and HCO3 in the opposite dir- the eel esophagus. As CLDNs are highly diversified in ection from that in SW eels [158]. Thus, the direction of 2− teleost fishes, the selectivity of CLDNs for ions and SO4 transport by SLC26a1 is concentration-dependent. water must be examined in diverse teleost CLDNs in the As Mg2+ is precipitated with Ca2+ as carbonates to de- 2− future. Hormonal regulation of paracellular transport is crease its concentration in the luminal fluid, SO4 may also an interesting topic for future studies. be absorbed to a greater extent than Mg2+. In other teleost fishes, cldn15 and cldn25b are Genes involved in Mg2+ transport are significantly expressed significantly, and their expression is upregu- expressed in the intestines of SW mefugu [96, 97]. In lated after SW transfer in the intestine of the Atlantic mammals, transient receptor potential melastatin types 6 salmon [235]. However, the expression of cldn3 is low in [252] and 7 [190], Cnnm4 [276], and Mg2+ transporter 1 this species [233]. The barrier-forming CLDN3 genes [65] are expressed in the intestine [41]. Furthermore, were duplicated and are expressed in different intestinal some TJ proteins, such as cation channel-type CLDN16 segments of the euryhaline pufferfish, Tetraodon nigro- and CLDN19, are known to selectively pass Mg2+ viridis; in addition, the expression levels decrease after through the paracellular route [118, 202]. As the Mg2+ transfer to SW [33]. The cldn15a, cldn15b and cldn25 concentration in the luminal fluid is extremely high (> are expressed in the medaka intestine, and cldn15b ex- 150 mM), the paracellular pathway needs to be taken pression is profoundly downregulated in SW [21]. As into account. However, expression of cldn16 and cldn19 the aqp genes are downregulated in the medaka intestine in the SW eel intestine is undetectable by RNA-seq. Al- after SW transfer [139], the decreased cldn15 expression though research on Mg2+ transport across the intestinal in SW may be related to increased water permeability of epithelium in teleost fishes is still in its infancy, this is the paracellular route (see 4.2.1). Recently, Tipsmark certainly an intriguing topic for future research. 2− et al. [234] suggested that CLDN15 in the medaka intes- Concerning SO4 transport, a pair of SLC26-family tine may be water-permeable because the density of im- transporters, apical SLC26a3/6 and basolateral SLC26a1, munoreactive CLDN15 at TJs did not change in SW are substantially expressed in the intestinal epithelia of despite downregulation of cldn15 transcripts. marine teleosts (see 4.4.1). These transporters have been − − suggested to absorb Cl and secrete HCO3 , as discussed 2+ 2− 6.2 Absorption of Mg and SO4 above [69]. We also suggest that SLC26a3/6 may ex- 2+ 2− It is generally accepted that Mg and SO4 are hardly change these monovalent ions in opposing directions, − − absorbed by the intestine in teleosts [166, 262]; thus, i.e., via Cl secretion and HCO3 absorption, as judged − − changes in their amounts and concentrations in the lu- by the high HCO3 concentration and low Cl concen- minal fluid are sometimes used as markers to estimate tration in the luminal fluid (see 4.3.1). In addition, we the amounts of ingested SW and subsequent water further suggest that this pair of SLC26 transporters may 2+ 2− − − absorption by the intestine, respectively. However, Mg absorb SO4 and secrete HCO3 or Cl (Figs. 5 and 6). 2− and SO4 may be absorbed significantly by the intestine This exchange is possible because of the high Takei Zoological Letters (2021) 7:10 Page 26 of 34

2− concentration of SO4 in the luminal fluid of the distal shown to be functionally coupled but not physically intestine (Table 1) and the high affinity of these SLC26 coupled with SLC26a3 [210]. 2− AEs for SO4 [1]. This anion exchange also increases The physical and functional association of SLC4-family − − luminal HCO3 and Cl concentrations to facilitate car- carbonate transporters with membrane-bound CAIV at bonate precipitation and to maintain NKCC2 activity for the extracellular domain of transporters to form metabo- water and NaCl absorption, respectively. It has been lons has also been reported [2, 209]. CAIV is anchored to 2− − shown that SO4 is absorbed in exchange for Cl in the the cell surface via a glycosylphosphatidylinositol domain 2− winter flounder intestine in vitro under high SO4 and and catalyzes mainly the reverse reaction to generate CO2 − 2− − + low Cl in the mucosal fluid, while SO4 secretion oc- and H2O from concentrated HCO3 and H in the micro- curs when both mucosal and serosal fluid contain 1 mM environment (Fig. 8). The generated CO2 can diffuse 2− 35 2− SO4 [169]. When SO4 is added to the environmen- across the lipid bilayer of the plasma membrane, but the 2− tal SW, 15% of the SO4 that enters the blood enters presence of AQP1 greatly accelerates its transport [171]. via intestinal absorption, as demonstrated by esophageal Water is also transported back into the cells by AQP1, 35 2− ligation in SW eels [257]. SO4 injected into the blood which is activated by CAII bound to the C-terminal 35 2− is secreted gradually into the environment; the SO4 DADD motif in the cytoplasmic domain of AQP1 [251]. appears in the urine as ions soon after injection, while it NHE3 and SLC26a3/6 have been suggested to interact is taken up by the liver and gills. Much more 35S than with CFTR for regulation of mutual activity in the mam- 35 2− SO4 is secreted slowly from the digestive tract and malian intestine [106] (Fig. 8). PDZ adaptor proteins 2− gills as bile compounds and mucus. Thus, more SO4 is such as NHERFs are scaffolding proteins that assemble absorbed by the intestine than is indicated by its urine membrane proteins with PDZ-binding motifs at the C- 2− concentration. Mefugu SLC26a6 can exchange SO4 terminal region [44, 123]. Such proteins include NHE3, − − with Cl /HCO3 , as demonstrated via transporter ex- SLC26-family transporters and CFTR. As NHERF1 ~ pression in Xenopus oocytes, and the activity is NHERF4 have multiple PDZ domains within their 2− dependent on the SO4 concentration in the medium structures, they assemble the several abovementioned 2− [107]. The ability of teleost SLC26a1 to exchange SO4 transporters and anchor them to the plasma membrane − − and Cl /HCO3 needs to be verified in a similar expres- (Fig. 8). They also assemble a number of kinases and 2− sion system in the future. The rate of SO4 absorption cytoskeletal molecules to form a multiprotein complex by the intestine also needs to be calculated using an in- that regulates transporter activity and recycling (traffick- testinal sac preparation containing different concentra- ing) of endocytic vesicles carrying transporters [123]. It 2− tions of SO4 in the lumen. is also suggested that the NHERF-based complex further polymerizes to form a complex metabolon for more ac- 6.3 Functional coupling of transporters and enzymes tive interaction among transporters. The C-terminal se- (metabolon formation) quences, including the PDZ-binding motif of CFTR, are It has been suggested that in mammals, NHEs, AEs, thought to determine its localization on the apical mem- CAII, and CAIVs form a metabolon on the apical brane [155, 217]. membrane in intestinal epithelial cells to facilitate the In intestinal epithelial cells, CFTR is coupled with + − + secretion of H and HCO3 and the absorption of Na SLC26 transporters through binding between its R- − and Cl across the apical membrane [199] (Fig. 8). Phys- domain and the STAS domains of SLC26 proteins on ical as well as functional association of NHE3 with CAII NHERFs [115, 128] (Fig. 8). The interaction of the two has been reported on the brush border membranes of transporters is usually mutually activated, resulting in in- + − − renal proximal tubules, where > 65% of filtered Na is creased HCO3 secretion or Cl secretion depending reabsorbed [120]. It has also been shown that CAII binds upon the tissues examined [106, 115, 212]. After en- to the C-terminal amino acid sequence (DADD) of AE1 dogenous cAMP stimulation via hormones, anion secre- − + (SLC4a1) and other SLC4-family HCO3 transporters tion is enhanced, while Na absorption is suppressed (AE2, AE3, and NBCe1) to form a complex called the (see 5). This is explained by inhibition of NHE3 by bicarbonate transport metabolon (Fig. 8), which maxi- CFTR bound to NHERF [32]. NHE3 has a PDZ-binding − mizes transmembrane HCO3 flux [152, 211]. In an- motif to bind to NHERFs but has no STAS domain to other group of AEs (the SLC26 family), SLC26a6 has bind with the CFTR R-domain. The interaction of NHE3 such a consensus DADD sequence at the C-terminal and SLC26a3 appears to be realized by the subcellular region for CAII binding, and both physical and func- linkage of two NHERFs to form a complex metabolon tional associations with the have been re- [124]. These data regarding the interaction were ob- ported (Fig. 8). The association is disrupted by Ang tained in a heterologous expression system using cul- II-induced PKC activation [3]. SLC26a3 does not pos- tured cells. More recently, however, mutual regulation of sess the consensus sequence, and CAII has been CFTR, SLC26a3/6 and NHE3 has been demonstrated Takei Zoological Letters (2021) 7:10 Page 27 of 34

in vivo in in situ perfused intestines using knockout suggests that the direction of trafficking is relatively flex- mice [204]. ible, but such flexibility for CFTR trafificking has been Based on the data from mammals mentioned above, a demonstrated only in fishes thus far. A small population − possible scheme for HCO3 secretion and carbonate for- of secretory-type cells exists among intestinal epithelial mation in the teleost intestine can be depicted (Fig. 8); cells in mammals; these cells express high levels of apical this scheme needs to be confirmed in the future. A membrane CFTR [99]. Prominent CFTR-tagged vesicles metabolon is proposed to exist on the apical membranes are also observed beneath the apical membrane. It seems of enterocytes that consists of NHE3, SLC26a3/6, and that cAMP stimulation recruits vesicles to the apical − CFTR assembled on the scaffolding protein NHERF via membrane and causes robust Cl and water secretion, their PDZ-binding motifs. The slc9a3r1 (NHERF1) is resulting in diarrhea. In contrast, the sorting of CFTR- expressed in the eel intestine and upregulated in the an- tagged vesicles seems to be more complex, being accom- terior intestine after SW acclimation [228]. Cytosolic plished not by cAMP alone but by cooperation with CAII and membrane-bound CAIV are connected to other messengers in the killifish intestine [145]. − transporter assembly for efficient conversion of HCO3 + NHEs are a group of transporters involved in intestinal + + + H ⇌ CO2 +H2O. AQP1 also exists near the metabolon Na absorption and H secretion [174]. Among the for quick recycling of CO2 and H2O across the apical NHEs, NHE1 and NHE2 are resident-type (nontraffick- membranes of enterocytes (Fig. 8). Grosell et al. [76] ing) transporters localized on the basolateral and apical suggested that VHA and SLC26a6 form a metabolon on membranes, respectively, in intestinal epithelial cells in the apical membrane of the marine teleost intestinal epi- mammals [280]. Thus, vesicles containing these NHEs − − thelium for active HCO3 secretion and Cl absorption. are tagged for trafficking and fusion with the plasma Thus, it is possible that VHA is also involved in this membrane soon after protein synthesis. On the other scheme to increase the efficiency of H+ acquisition and hand, NHE3-containing cytoplasmic vesicles are tagged to further accelerate the conversion by CAIV and Mg/ and inserted into the apical plasma membrane upon CaCO3 formation. The whole scheme needs to be eluci- stimulation by intracellular messengers [280]. Since dated in teleosts in the future. nhe2 and nhe3 are expressed in significant amounts in the esophagus and intestine in eels and other 6.4 Trafficking of transporter-tagged cytoplasmic vesicles teleosts, it would be interesting to investigate whether It is well known that AQP2 is stored on endocytic vesicles a similar difference in the regulation of trafficking in renal collecting duct cells and inserted into the apical also exists among NHEs in teleost fishes. membrane upon stimulation by vasopressin-induced cAMP production in mammals [102]. This recruitment enables quick regulation of water reabsorption by vaso- Conclusions pressin to cope with acute dehydration. Conversely, AQP2 on the plasma membrane is internalized via endocytic ves- 1. Anguillid eels are extraordinarily euryhaline teleosts icles that are stored in the subapical epithelial cell layer and offer many advantages for research on the role for future recruitment. This phenomenon is called vesicle of the digestive tract in SW acclimation. Although trafficking and is achieved by phosphorylation of trans- eels are now endangered species, research in porters and activation of cytoskeletal molecules. In the cultured eels will enable great progress to be made case of AQP2, PKA is responsible for initial phosphoryl- toward understanding the mechanisms of SW ation. On the other hand, the scaffolding protein NHERF acclimation in the future. with PDZ domains is connected to the actin cytoskeleton 2. The key processes in eels (and marine teleosts) that by linker proteins in the Ezrin/Radixin/Moesin (ERM) enable water gain after SW drinking are protein family and the protein complex with transporters desalinization by the esophagus without osmotic mediates vesicle trafficking [129, 196]. Therefore, trans- water loss, bicarbonate secretion for Mg/CaCO3 porters with PDF-binding motifs such as CFTR, NHE3, precipitation in the intestine, and efficient water/ and SLC26a6 are likely to be under the control of vesicle NaCl absorption by the intestine using unique trafficking (Fig. 8). transporters. Vesicle trafficking also seems to occur in teleost fishes, 3. The major transporters responsible for such processes and the direction of trafficking is dependent upon the have been illuminated in eels and other marine osmotic environment. For instance, CFTR-tagged vesi- teleosts. As transcriptomic data on the digestive tracts − cles are recruited to the basolateral membrane for Cl of eels after transfer from FW to SW are available to absorption when killifish are in FW, while the vesicles the public (DDBJ accession number: DRA004258), − are recruited to the apical membrane for Cl secretion genes that are involved in fine-tuning of transporter after acclimation to SW, as mentioned above [145]. This function will be elucidated in the near future. Takei Zoological Letters (2021) 7:10 Page 28 of 34

4. Several biological processes in the digestive tract Competing interests that have not received sufficient attention but need The author declares that he has no competing interests. to be investigated are suggested for future studies. Received: 2 September 2020 Accepted: 16 April 2021

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